JP6985153B2 - Novel peptides and peptides and their scaffolds for use in immunotherapy for colorectal cancer (CRC) and other cancers - Google Patents

Description

The present invention relates to peptides, proteins, nucleic acids, and cells for use in immunotherapy. In particular, the present invention relates to immunotherapy for cancer. The invention further relates to tumor-related T cell peptide epitopes, alone or in combination with other tumor-related peptides, which may, for example, stimulate an antitumor immune response or stimulate T cells in vitro to a patient. Can serve as the active pharmaceutical ingredient of the vaccine composition to be transferred to. Peptides that bind to major histocompatibility complex (MHC) molecules, or the peptides themselves, can also be targets for antibodies, soluble T cell receptors, and other binding molecules.

The present invention relates to several novel peptide sequences and variants thereof derived from HLA class I molecules of human tumor cells, which are in vaccine compositions for eliciting an antitumor immune response, or pharmacologically / immune. It can be used as a target for the development of immunologically active compounds and cells.

Colorectal cancer (CRC) is the third most common cancer in men and the second most common cancer in females. Worldwide, CRC accounts for about 10% of all newly diagnosed cancer cases. In 2012, 746,000 males and 614,000 females were diagnosed with 1.36 million new CRC cases, resulting in a male-to-female ratio of 1.2: 1 (World Cancer Report, 2014). .. CRC is a disease of the elderly. The average age at diagnosis is 68 years (SEER STAT facts, 2014).

Incidences vary geographically by about 10-fold and are similar in males and females. The highest incidence in both men and women is found in Australia / New Zealand (age-adjusted prevalence (ASR) per 100,000 males (ASR) = 45, ASR per 100,000 females = 32). The incidence in Europe varies slightly, with ASR = 38 per 100,000 males and ASR = 25 per 100,000 females. The lowest incidence in the world is found in West Africa, with 4.5 per 100,000 males and 3.8 per 100,000 females (World Cancer Report, 2014).

The overall 5-year survival rate from CRC is about 65%. However, survival depends on the stage at diagnosis. The 5-year survival rate for localized CRC is 89.8%, and for regional and remote CRC it is 70.5% and 12.9%, respectively. CRC is the fourth highest cause of cancer deaths (694,000 deaths; 8.5%) (SEER Stat facts, 2014; World Cancer Report, 2014).

CRC is usually diagnosed using a TNM system that incorporates information about the size of the primary tumor (T), lymph node involvement (N), and the development of distant metastases (M). The UICC (Union International Control Center) progression classification is based on the TNM system and includes statistical data for prognostic prediction (Stinzing, 2014).

Risk factors for developing CRC include lifestyle-related factors, hereditary predisposition, and inflammatory conditions. Excessive alcohol use, smoking, and obesity are associated with an increased risk of developing CRC. Hereditary risk factors include familial onset of CRC, familial adenomatous polyposis (FAP), attenuated FAP (AFAP) / attenuated colorectal adenomatous polyposis (AAPC), and hereditary nonpolyposis colorectal cancer (AAPC). HNPCC), and adenomatous polyp syndrome. Inflammatory conditions associated with increased risk of CRC include inflammatory bowel disease (IBD) such as ulcerative colitis and Crohn's disease (Baena and Salinas, 2015; Stintzing, 2014; Vasen et al., 2015).

Histologically, more than 90% of all CRCs are glandular cancers. Rare CRC types include neuroendocrine, squamous cells, adenosquamous epithelium, spindle cells, and undifferentiated carcinoma (Fleming et al., 2012). The majority of colorectal adenocarcinomas result from adenomas or dysplastic precursor lesions. Different molecular mechanisms contribute to tumorigenesis, depending on the type of lesion / carcinoma. The chromosomal instability (CIN) pathway (the "suppressor" pathway) is characterized by mutations in the APC, KRAS or p53 genes. Additional mutations are found in the LKB1 / STK11, SMAD4, BMPR1A or MYH genes. The microsatellite instability (MSI) pathway (the "mutator" pathway) comprises mutations in the DNA mismatch repair (MMR) genes MLH1, MSH2, MSH6, and PMS2, overmethylation of the MMR gene, or BRAF mutations. .. Epigenetic instability, including DNA methylation, histone alterations, and chromatin reconstitution, is characteristic of CIMP (CpG island methylator phenotype) tumors (Fleming et al., 2012).

Different standard treatments are available for colorectal and rectal cancer, depending on the stage of the CRC. Standard procedures include surgery, radiation therapy, chemotherapy, and CRC targeted therapy (Berman et al., 2015a; Berman et al., 2015b).

Tumor removal is essential for the treatment of CRC. The anatomical conditions of rectal cancer differ from other CRCs because the rectum is located within the pelvis and the tumor can be difficult to access. Well-differentiated small rectal tumors (stage T1) require resection but do not require further treatment with chemotherapy. Patients with higher stage T rectal tumors receive neoadjuvant radiochemotherapy with fluoropyrimidine prior to total colonic rectotomy (TME) and adjuvant chemotherapy. In chemotherapeutic treatment, the drug capecitabine or 5-fluorouracil (5-FU) is used. For combination chemotherapy, a cocktail containing 5-FU, leucovorin, and oxaliplatin (FOLFOX) is recommended (Stintzing, 2014; Berman et al., 2015b).

Treatment of colon cancer involves radical colon resection and lymphadenectomy. Early stage (UICC stage I) does not require additional treatment. Patients with UICC stage II tumors receive 5-FU or capecitabine. Treatment of patients with UICC stage III includes a combination of FOLFOX and XELOX (capecitabine + oxaliplatin) (Berman et al., 2015a; Stintzing, 2014).

Metastatic and unresectable CRCs are FOLFIRI (5-FU, leucovorin, irinotecan), FOLFOX, FOLFOXIRI (5-FU, irinotecan, oxaliplatin), FOLFOX / capesitabin, FOLFOX / oxaliplatin, FOLFOX / oxaliplatin, FOLFIR. Treated with chemotherapeutic cocktails such as (5-FU, Tegafur / uracil) (Stintzing, 2014).

In addition to chemotherapeutic agents, some monoclonal antibodies that target epidermal growth factor receptors (EGFR, cetuximab, panitumab) or target vascular endothelial growth factor A (VEGF-A, bevacizumab) are high. It is administered to patients with stage disease. For second-line and late-stage treatments, the VEGF inhibitor aflibercept, the tyrosine kinase inhibitor regorafenib, and the thymidylate synthesizer inhibitor TAS-102, and the dUTPase inhibitor TAS-114 can be used (Stintzing, 2014). Wilson et al., 2014).

The latest clinical trials analyze active immunotherapy as a treatment option for CRC. These strategies include tumor-related antigen (TAA) -derived peptides, whole tumor cells, dendritic cell (DC) vaccines, and vaccination with viral vectors (Koido et al., 2013).

Peptide vaccines have previously included carcinoembryonic antigen (CEA), mutin 1, EGFR, squamous cell carcinoma antigen recognized by T cell 3 (SART3), β-human chorionic gonadotropin (β-hCG), It is directed to the translocase of Wilms tumor antigen 1 (WT1), survivin-2B, MAGE3, p53, ringfinger protein 43, and mitochondrial outer membrane 34 (TOMM34), or mutant KRAS. In several Phase I and II clinical trials, patients showed antigen-specific CTL response or antibody production. In contrast to the immunological response, many patients did not benefit from the peptide vaccine at the clinical level (Koido et al., 2013; Miyagi et al., 2001; Moulton et al., 2002; Okuno et al., 2011).

Dendritic cell vaccines comprises DCs pulsed with either TAA-derived peptides, tumor cell lysates, apoptotic tumor cells, or tumor RNA or DC tumor cell fusion products. Many patients in Phase I / II trials showed specific immunological responses, while only a few had clinical benefits (Koido et al., 2013).

Whole tumor cell vaccines consist of irradiation-modified autologous tumor cells or virus-infected irradiated cells modified to secrete GM-CSF. Most patients showed no clinical benefit in some Phase II / III trials (Koido et al., 2013).

Vaccinia virus or replication defective avian poxvirus encoding CEA, as well as B7.1, ICAM-1, and LFA-3 have been used as vehicles for viral vector vaccines in Phase I clinical trials. In another study, a non-replicating canary poxvirus encoding CEA and B7.1 was used. In addition to inducing a CEA-specific T cell response, 40% of patients showed an objective clinical response (Horizon et al., 2000; Kaufman et al., 2008).

Given the severe side effects and costs associated with treating cancer, it is necessary to identify factors that can be used to treat cancer in general, and in particular CRC. There is also a need to identify biomarker-equivalent factors for cancer in general, especially CRC, that lead to better diagnosis of cancer, assessment of prognosis, and prediction of successful treatment.

Immunotherapy for cancer represents an option to minimize side effects while specifically targeting cancer cells. Cancer immunotherapy takes advantage of the presence of tumor-related antigens.

The current classification of tumor-related antigens (TAA) consists of the following major groups:
a) Cancer testis antigen: The first TAA identified so far that can be recognized by T cells belongs to this class and was originally referred to as the cancer testis (CT) antigen, but its members are tissues. This was because it was expressed in histologically different human tumors and was present only in testicular spermatocytes / spermatogonia in normal tissues, sometimes in the placenta. Since testis cells do not express class I and II HLA molecules, these antigens cannot be recognized by normal tissue T cells and are therefore immunologically considered tumor specific. Well-known examples of CT antigens are MAGE family members and NY-ESO-1.
b) Differentiation antigens: These TAAs are shared between the tumor and the normal tissue from which it develops. Most of the known differentiation antigens are found in melanoma and normal melanocytes. Many of these melanocyte-related proteins are involved in melanin biosynthesis and are therefore not tumor-specific, but are still widely used for cancer immunotherapy. Examples include, but are not limited to, tyrosinase and Melan-A / MART-1 for melanoma, or PSA for prostate cancer.
c) Overexpressed TAA: Genes that encode widely expressed TAA have been detected in histologically different types of tumors and generally at lower expression levels in many normal tissues. While many of the epitopes that can be processed and potentially presented by normal tissue can be below threshold levels for T cell recognition, their overexpression in tumor cells by disrupting previously established immune tolerance. , Can initiate an anti-cancer response. Prominent examples of this class of TAA are Her-2 / neu, survivin, telomerase or WT1.
d) Tumor-specific antigens: These unique TAAs result from mutations in normal genes (β-catenin, CDK4, etc.). Some of these molecular changes are associated with neoplastic transformation and / or progression. Tumor-specific antigens can usually induce a strong immune response without the risk of an autoimmune response to normal tissue. On the other hand, these TAAs are most often associated only with the very tumor on which they are identified and are usually not shared among many individual tumors. For proteins with tumor-specific (associative) isotypes, tumor specificity (or association) of the peptide may also occur if the peptide is derived from a tumor (related) exon.
e) TAA resulting from aberrant post-translational modifications: Such TAA may result from proteins that are not specific and are not overexpressed in the tumor, but nonetheless, the tumor is predominantly active in the tumor by a post-translational process. Become relevant. Examples of this class result from modified glycosylation patterns that result in novel epitopes such as MUC1 in tumors, or events such as protein splicing during degradation that may or may not be tumor specific.
f) Oncoviral proteins: These TAAs are viral proteins, which may play important roles in the carcinogenic process and are exogenous (not of human origin), so they can elicit a T cell response. Examples of such proteins are human papilloma type 16 viral proteins E6 and E7 expressed in cervical cancer.

T cell-based immunotherapy targets tumor-related or tumor-specific protein-derived peptide epitopes presented by major histocompatibility complex (MHC) molecules. Antigens recognized by tumor-specific T lymphocytes, ie their epitopes, can be molecules derived from all protein classes such as enzymes, receptors, transcription factors, etc., and they are expressed in each tumor cell. , Usually upregulated compared to unmodified cells of the same origin.

There are two classes of MHC molecules, MHC class I and MHC class II. The MHC class I molecule is composed of α heavy chain and β2 microglobulin, and the MHC class II molecule is composed of α and β chain. Their three-dimensional structure provides a binding groove used for non-covalent interactions with peptides.

MHC class I molecules are found on most nucleated cells. They present peptides primarily obtained from protein cleavage of endogenous proteins, defective ribosome products (DRIPs), and larger peptides. However, peptides from the endosomal compartment or extrinsic origin are also frequently found on MHC class I molecules. This non-classical form of Class I presentation is referred to in the literature as cross-presentation (Brossart and Bevan, 1997; Rock et al., 1990). MHC class II molecules are predominantly found in professional antigen presenting cells (APCs), eg, peptides of exogenous or transmembrane proteins that are taken up by APCs and subsequently processed during endocytosis. ..

The peptide-MHC class I complex is recognized by CD8-positive T cells with the appropriate T cell receptor (TCR), while the peptide-MHC class II molecule complex is CD4 positive with the appropriate TCR. Recognized by helper T cells. As a result, it is well known that TCRs, peptides, and MHC are stoichiometrically present in 1: 1: 1 amounts.

CD4-positive helper T cells play an important role in inducing and maintaining an effective response by CD8-positive cytotoxic T cells. Identification of CD4-positive T cell epitopes derived from tumor-related antigens (TAAs) is crucial for the development of drugs that initiate an antitumor immune response (Gnjatic et al., 2003). At the tumor site, T helper cells maintain a cytotoxic T cell (CTL) -friendly cytokine environment (Mortara et al., 2006), eg, CTL, natural killer (NK) cells, macrophages, and granulocytes. Attracts effector cells of (Hwang et al., 2007).

In the absence of inflammation, expression of MHC class II molecules is primarily limited to immune system cells, particularly professional antigen presenting cells (APCs) such as monocytes, monocyte-derived cells, macrophages, dendritic cells and the like. Tumor cells have been shown to express MHC class II molecules in cancer patients (Dengjel et al., 2006).

The extended (longer) peptide of the invention can act as an MHC class II active epitope.

T helper cells activated by MHC class II epitopes play an important role in integrating the effector function of CTL in antitumor immunity. T-helper cell epitopes that initiate TH1-type T-helper cell responses are CD8-positive killer T cells directed at tumor cells that present tumor-related peptide / MHC complexes on their cell surface, including cytotoxic function. Supports the effector function of cells. In this way, the tumor-related T-helper cell peptide epitope can serve as an active pharmaceutical component of a vaccine composition that stimulates an antitumor immune response, either alone or in combination with other tumor-related peptides.

For example, in mammalian models such as mice, CD4-positive T cells are sufficient to inhibit tumor development through inhibition of angiogenesis by the secretion of interferon gamma (IFNγ), even in the absence of CD8-positive T lymphocytes. It was shown to be (Beaty and Patterson, 2001; Mumberg et al., 1999). There is evidence that CD4 T cells are a direct antitumor effector (Braumuller et al., 2013; Trans et al., 2014).

Since the constitutive expression of HLA class II molecules is usually limited to immune cells, it has not previously been considered possible to isolate class II peptides directly from primary tumors. However, Dengjell et al. Succeededly identified several MHC class II epitopes directly from tumors (International Publication No. 2007/028574, European Patent No. 1760888B1).

CD8 + T cells (ligand: MHC class I molecule + peptide epitope) or CD4 positive T helper cells (ligand: ligand:) because both CD8 and CD4 dependent types of responses jointly contribute to antitumor effects. Identification and characterization of tumor-related antigens recognized by either MHC class II molecules + peptide epitopes) is important for the development of tumor vaccines.

In order for an MHC class I peptide to initiate (trigger) a cell-mediated immune response, it must also bind to MHC molecules. This process depends on the alleles of the MHC molecule and the specific polymorphism of the amino acid sequence of the peptide. MHC class I-binding peptides are usually 8-12 amino acid residue lengths and usually have two conserved residues (“anchors”) in their sequence that interact with the corresponding binding groove of the MHC molecule. contains. In this way, each MHC allele has a "binding motif" that determines which peptide can specifically bind to the binding groove.

In an MHC class I-dependent immune response, peptides can not only bind to specific MHC class I molecules expressed by tumor cells, but they are also subsequently recognized by T cells with specific T cell receptors (TCRs). Must be done.

Certain requirements must be met in order for a protein to be recognized by T lymphocytes as a tumor-specific or tumor-related antigen and used therapeutically. The antigen should be expressed primarily by tumor cells, not by healthy tissue, or expressed in relatively small amounts. In a preferred embodiment, the peptide should be over-presented by the tumor cells as compared to healthy tissue. It is even more desirable that each antigen is present not only in certain tumors, but also in high concentrations (ie, the number of copies per peptide cell). Tumor-specific and tumor-related antigens are often derived from proteins that are directly involved in the transformation of normal cells into tumor cells, for example because of their function in cell cycle regulation or apoptosis suppression. In addition, downstream targets of proteins that are directly responsible for transformation may be upregulated and thus indirectly tumor-related. Such indirect tumor-related antigens may also be targets for vaccination approaches (Singh-Jasuja et al., 2004). Epitopes are present in the amino acid sequence of the antigen to ensure that such peptides (“immunogenic peptides”) are derived from tumor-related antigens and result in in vitro or in vivo T cell responses. It is essential to do.

Basically, any peptide that can bind to MHC molecules may function as a T cell epitope. A requirement for in vitro or in vivo T cell response induction is the presence of T cells with the corresponding TCR and the absence of immune tolerance to this particular epitope.

Therefore, TAA is a starting point for the development of T cell-based therapies, including but not limited to tumor vaccines. Methods for identifying and characterizing TAA are usually based on the use of T cells that can be isolated from patients or healthy individuals, or they have differential transcriptional profiles between tumors and normal tissues, or differential peptide expression. Based on pattern generation. However, identification of genes that are overexpressed in tumor tissue or human tumor cell lines, or selectively expressed in such tissues or cell lines, is accurate with respect to the use of antigens transcribed from these genes in immunotherapy. Do not provide any information. This is because only individual subpoples of the epitopes of these antigens are suitable for such applications, because T cells with the corresponding TCR must be present and this particular epitope. This is because the immune tolerance to the disease must be absent or minimal. Therefore, in a highly preferred embodiment of the invention, it is important to select only peptides that are presented in excess or selectively in which functional and / or proliferative T cells are found. Such functional T cells are defined as T cells (“effector T cells”) that can be cloned and proliferated upon stimulation with a specific antigen and can perform effector function.

When the peptide MHC is targeted by a specific TCR (eg, soluble TCR) according to the invention and an antibody or other binding molecule (scaffold), the immunogenicity of the underlying peptide is secondary. In these cases, presentation is the deciding factor.

In a first aspect of the invention, the invention is at least 77%, preferably at least 88%, homologous (preferably at least 77% or at least) with SEQ ID NO: 1 to SEQ ID NO: 191 or SEQ ID NO: 1 to SEQ ID NO: 191. With respect to a peptide comprising an amino acid sequence selected from the group consisting of the mutant sequence (88% identical), the variant in which the variant binds to MHC and / or T cells and the peptide or pharmaceutically thereof. It induces cross-reactivity with acceptable salts, in which the peptide is not the underlying full-length polypeptide.

The present invention is from a variant thereof that is at least 77%, preferably at least 88% homologous (preferably at least 77% or at least 88% identical) to SEQ ID NO: 1 to SEQ ID NO: 191 or SEQ ID NO: 1 to SEQ ID NO: 191. With respect to the peptides of the invention comprising sequences selected from the group, said peptides or variants thereof have a full length of 8-100, preferably 8-30, most preferably 8-14 amino acids.

The following table shows the peptides according to the invention, their respective SEQ ID NOs, and the predicted origin (basic) genes of those peptides. All peptides in Tables 1 and 2 bind to ^{HLA-A * 02.} The peptides in Table 2 have been previously disclosed in a large list as a result of high-throughput screening with high error rates or calculated using algorithms, but have not been associated with cancer at all so far. rice field. The peptides in Table 3 are additional peptides that may be useful in combination with other peptides of the invention. The peptides of Table 4A and B are further useful in the diagnosis and / or treatment of various other malignancies with overexpression or overpresentation of their respective basal polypeptides.

The present invention further relates to, for example, lung cancer, brain cancer, stomach cancer, kidney cancer, liver cancer, pancreatic cancer, prostate cancer, leukemia, breast cancer, merkel cell cancer (MCC), melanoma, ovarian cancer, and the like. And the peptides according to the invention for use in the treatment of proliferative disorders such as esophageal cancer.

Particularly preferred are the single or combined peptides of the invention selected from the group consisting of SEQ ID NOs: 1 to SEQ ID NO: 191. More preferably, a single or combination peptide selected from the group consisting of SEQ ID NOs: 1 to SEQ ID NO: 68 (see Table 1) and CRC, lung cancer, brain cancer, gastric cancer, kidney cancer, liver. Cancer, pancreatic cancer, prostate cancer, leukemia, breast cancer, merkel cell carcinoma, melanoma, ovarian cancer, and esophageal cancer, and preferably their use in immunotherapy of CRC.

Most preferred are SEQ ID NOs: 1, 3, 6, 11, 13, 16, 18, 19, 22, 23, 24, 26, 31, 32, 34, 37, 40, 44, 45, 59, 67, 71. , 82, 87, 88, 100, 103, 105, 113, 123, 124, 126, 129, 131, 132, 133, 135, 137, 140, 142, 150, 152, 153, and SEQ ID NO: 166. Single or combination peptides selected from, and CRC, lung cancer, brain cancer, stomach cancer, kidney cancer, liver cancer, pancreatic cancer, prostate cancer, leukemia, breast cancer, merkel cell cancer, melanoma, ovary Cancer, and esophageal cancer, and preferably their use in immunotherapy of CRC. The peptide of SEQ ID NO: 22 is also particularly preferred in the method of making each T cell receptor (TCR) described below.

As shown in Tables 4A and B below, many of the peptides according to the invention are also found on other tumor types and can therefore be used in immunotherapy for other indications. See also FIG. 1D and Example 1.

Table 4A: Peptides according to the invention, and their specific use in other proliferative disorders, especially other cancerous disorders. The table is presented with a geometric mean ratio of tumor to normal tissue greater than 3 for selected peptides, either over 5% of the measured tumor samples or over 5% of the measured tumor samples. Either they are, they indicate the additional tumor type found on it. Overpresentation is defined as presentation on a higher tumor sample compared to a normal sample with maximum presentation.

Table 4B: Peptides according to the invention, and their specific use in other proliferative disorders, especially other cancerous disorders (amendments to Table 4A). The table shows overpresentation of the selected peptide in more than 5% of the measured tumor samples, or more than 3 tumors vs. normal tissues in more than 5% of the measured tumor samples, as shown in Table 4A. Presented in geometric mean ratios, they indicate the additional tumor types found on it. Overpresentation is defined as presentation on a higher tumor sample compared to a normal sample with maximum presentation. Normal tissues tested for overpresentation are adipose tissue, adrenal glands, blood cells, blood vessels, bone marrow, brain, esophagus, eye, gallbladder, heart, kidney, large intestine, liver, lungs, lymph nodes, nerves, pancreas, vices. The thyroid gland, peritoneum, pituitary gland, thoracic membrane, salivary gland, skeletal muscle, skin, small intestine, spleen, stomach, thyroid gland, trachea, urinary tract, and bladder.

Accordingly, another aspect of the invention is, in one preferred embodiment, SEQ ID NOs: 1, 3, 4, 6, 11, 15, 26, 31, 40, 45, 56, 59, for combination therapy for lung cancer. 62, 72, 77, 79, 84, 87, 88, 90, 102, 107, 110, 117, 123, 124, 125, 126, 128, 131, 132, 133, 134, 135, 139, 140, 141, 142, 143, 151, 152, 153, 154, 156, 157, 160, 161, 165, 167, 168, 170, 178, 184, 190, 192, 194, 203, 204, 205, 206, 208, 210, 212, 214, 215, 216, 217, 218, 222, 223, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241. 242, 243, 244, 245, 246, 247, 248, 250, 252, 255, 256, 257, 259, and 262, which relates to the use of at least one of the peptides according to the invention.

Accordingly, another aspect of the invention is, in one preferred embodiment, SEQ ID NOs: 1, 11, 19, 31, 44, 45, 58, 63, 72, 103, 108, for combination therapy of brain cancer. 118, 125, 126, 128, 131, 132, 133, 137, 157, 158, 162, 163, 164, 169, 178, 180, 181, 191, 194, 201, 205, 207, 208, 209, 210, At least one of the peptides according to the invention according to any one of 216, 217, 221, 222, 227, 230, 231, 234, 237, 240, 241 and 242, 243, 244, 247, 248, and 250. Regarding use.

Accordingly, another aspect of the invention is, in a preferred embodiment, any one of SEQ ID NOs: 65, 70, 133, 146, 225, 226, 230, 237, 252, and 256 for combination therapy with gastric cancer. With respect to the use of at least one of the peptides according to the invention described in one.

Accordingly, another aspect of the invention is, in one preferred embodiment, SEQ ID NOs: 31, 45, 48, 53, 56, 61, 70, 72, 83, 88, 102, for combination therapy for kidney cancer. 105, 108, 117, 123, 129, 133, 141, 142, 146, 147, 152, 157, 158, 160, 168, 170, 172, 184, 194, 199, 203, 205, 206, 209, 210, 211,217,218,221,222,225,227,232,233,234,235,240,241,242,243,244,245,246,247,250,251,259,261,264, and 265 With respect to the use of at least one of the peptides according to the invention according to any one of the above.

Accordingly, another aspect of the invention is, in one preferred embodiment, SEQ ID NOs: 6, 10, 26, 31, 33, 45, 47, 54, 56, 59, 62, for combination therapy of liver cancer. 63, 71, 72, 76, 79, 84, 88, 89, 98, 103, 105, 108, 117, 121, 123, 124, 126, 131, 132, 133, 134, 136, 139, 142, 148, 153, 154, 156, 157, 158, 159, 161, 166, 168, 169, 170, 171, 174, 178, 181, 184, 191, 193, 194, 195, 204, 205, 206, 207, 208, 209, 210, 214, 216, 217, 220, 221, 222, 225, 227, 229, 230, 231, 232, 234, 235, 237, 238, 239, 240, 241, 242, 243, 244, 245, With respect to the use of at least one of the peptides according to the invention according to any one of 250, 252, 253, 254, 259, 260, 261 and 264.

Accordingly, another aspect of the invention is, in one preferred embodiment, SEQ ID NOs: 3, 8, 38, 40, 44, 53, 55, 110, 116, 117, 146, for combination therapy of pancreatic cancer. 152, 170, 184, 198, 201, 203, 205, 209, 212, 224, 226, 227, 230, 232, 233, 236, 240, 241 and 242, 245, 246, 247, 248, 251 and 252, The use of at least one of the peptides according to the invention according to any one of 255, 256, 259, 261 and 262.

Accordingly, another aspect of the invention is, in one preferred embodiment, SEQ ID NOs: 25, 45, 48, 63, 69, 70, 101, 103, 116, 131, 178, for combination therapy of prostate cancer. At least one of the peptides according to the invention according to any one of 183, 184, 187, 204, 205, 206, 214, 216, 220, 221, 230, 231, 234, 243, 247, 257, and 259. Regarding use.

Accordingly, another aspect of the invention is, in one preferred embodiment, SEQ ID NOs: 6, 8, 29, 31, 58, 67, 72, 80, 82, 83, 110, 118, for combination therapy of leukemia. 121, 129, 149, 153, 154, 160, 161, 169, 171, 173, 180, 185, 199, 221, 227, 228, 239, 242, and 247. With respect to at least one use of.

Accordingly, another embodiment of the invention, in a preferred embodiment, is SEQ ID NO: 3, 45, 72, 77, 91, 116, 128, 133, 146, 161, 180, 183, for combination therapy of breast cancer. 188, 200, 214, 226, 230, 232, 233, 242, 245, 246, 252, and 258, relating to the use of at least one of the peptides according to the invention according to any one of the following.

Accordingly, another embodiment of the invention, in a preferred embodiment, is SEQ ID NO: 33, 83, 123, 124, 128, 133, 171 and 184, 197, 231, 237 for combination therapy with Merkel cell carcinoma. The use of at least one of the peptides according to the invention according to any one of 249, 250, and 257.

Accordingly, another aspect of the invention is, in one preferred embodiment, SEQ ID NOs: 6, 13, 58, 62, 67, 83, 88, 92, 118, 124, 125, 134, for combination therapy with melanoma. 139, 140, 142, 145, 149, 153, 156, 159, 161, 167, 170, 172, 174, 177, 178, 184, 185, 188, 199, 202, 216, 225, 236, 238, 242, The use of at least one of the peptides according to the invention according to any one of 244, 252, and 259.

Accordingly, another aspect of the invention is, in one preferred embodiment, SEQ ID NOs: 1, 3, 4, 6, 16, 18, 25, 31, 34, 44, 45, for combination therapy with ovarian cancer. 54, 57, 63, 72, 74, 87, 88, 91, 102, 108, 116, 123, 124, 125, 126, 127, 128, 131, 132, 133, 134, 137, 139, 140, 142, 146, 152, 153, 154, 159, 160, 161, 165, 167, 172, 174, 175, 177, 178, 181, 184, 185, 189, 193, 194, 195, 202, 203, 204, 205, 206, 208, 214, 215, 216, 217, 225, 226, 227, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 243, 246, 249, 252, 255, 256, 257, 258, 259, 261 and 265, according to any one of the following relates to the use of at least one of the peptides according to the invention.

Accordingly, another aspect of the invention is, in one preferred embodiment, SEQ ID NOs: 1, 3, 11, 15, 19, 26, 31, 37, 40, 46, 54, for combination therapy with esophageal cancer. 59, 62, 63, 65, 70, 72, 81, 82, 83, 84, 88, 89, 90, 98, 102, 117, 119, 123, 124, 125, 126, 128, 132, 133, 134, 138, 139, 141, 142, 144, 152, 153, 154, 155, 157, 158, 162, 169, 170, 177, 178, 184, 186, 192, 194, 197, 200, 202, 205, 210, 212, 214, 215, 216, 217, 225, 226, 227, 230, 232, 233, 234, 235, 236, 237, 238, 239, 241, 245, 246, 247, 248, 249, 250, 251, 252, 255, 259, 261 and 265, relating to the use of at least one of the peptides according to the invention.

Therefore, another aspect of the invention is preferably CRC, lung cancer, brain cancer, gastric cancer, kidney cancer, liver cancer, pancreatic cancer, prostate cancer, leukemia, breast cancer, merkel cell cancer, melanoma, Concerning the use of peptides according to the invention for the combined treatment of proliferative disorders selected from the group of ovarian cancer and esophageal cancer.

The present invention further relates to peptides according to the invention, which have the ability to bind to human major histocompatibility complex (MHC) class I molecules, or to MHC class II in extended form such as length variants.

The present invention further relates to the peptides according to the invention, wherein the peptides consist of, or essentially consist of, the amino acid sequences set forth in (respectively) SEQ ID NOs: 1 to SEQ ID NO: 191.

The invention further relates to peptides according to the invention, wherein the peptide comprises a modified and / or non-peptide bond.

The present invention further relates to the peptide according to the invention, wherein the peptide is particularly an antibody such as an antibody fused to the N-terminal amino acid of an HLA-DR antigen-related invariant chain (Ii) or specific to a dendritic cell, for example. Part of a fusion protein fused to (or in its sequence).

The invention further relates to nucleic acids encoding peptides according to the invention. The present invention further relates to nucleic acids according to the invention, which are DNA, cDNA, PNA, RNA, or combinations thereof.

The invention further relates to an expression vector capable of expressing and / or expressing a nucleic acid according to the invention.

The invention further relates to peptides according to the invention, nucleic acids according to the invention or expression vectors according to the invention for use in the treatment of diseases and in medicine, especially in the treatment of cancer.

The present invention further relates to antibodies that specifically oppose the peptide according to the invention or the complex of the peptide according to the invention and the MHC, and methods for producing them.

The present invention has T cell receptors (TCRs), particularly soluble TCRs (sTCRs), and cloned TCRs, and methods for producing them, as well as said TCRs, which are self-derived or integrated into allogeneic T cells. Further relating to the method of producing NK cells or other cells that cross-react with the TCR.

Antibodies and TCRs are additional embodiments of the immunotherapeutic use of peptides according to the invention.

The invention further relates to a host cell comprising the nucleic acid or expression vector according to the invention as described above. The present invention further relates to host cells according to the invention, which are antigen presenting cells, preferably dendritic cells.

The present invention further relates to a method for producing a peptide according to the invention, comprising the steps of culturing a host cell according to the invention and isolating the peptide from the host cell or a culture medium thereof.

INDUSTRIAL APPLICABILITY The present invention comprises contacting an antigen-presenting cell with a sufficient amount of antigen to load the antigen onto a class I or II MHC molecule expressed on the surface of a suitable antigen-presenting cell or artificial antigen-presenting cell. Further relating to the method according to the invention.

The present invention comprises an expression vector in which an antigen presenting cell can or expresses said peptide containing SEQ ID NOs: 1-SEQ ID NO: 191, preferably SEQ ID NO: 1-SEQ ID NO: 68, or a mutant amino acid sequence. Further relating to the method according to the present invention.

The present invention further relates to activated T cells produced by the method according to the invention, wherein the T cells selectively recognize cells expressing a polypeptide comprising the amino acid sequence according to the invention.

The present invention comprises target cells that abnormally express a polypeptide comprising any amino acid sequence according to the invention in a patient, comprising the step of administering to the patient an effective number of T cells produced according to the invention. More on how to kill.

The invention is the invention of any peptide described, nucleic acid according to the invention, expression vector according to the invention, cells according to the invention, activated T lymphocytes according to the invention, T cell receptors or in the manufacture of a drug. Further relating to the use of antibodies or other peptide-and / or peptide-MHC-binding molecules. Preferably, the agent is effective against cancer.

Preferably, the agent is a cell therapy, vaccine or protein based on soluble TCR or antibody.

The present invention further relates to the use according to the present invention, wherein the cancer cells are CRC, lung cancer, brain cancer, gastric cancer, kidney cancer, liver cancer, pancreatic cancer, prostate cancer, leukemia, breast cancer, merkel cells. Cancer (MCC), melanoma, ovarian cancer, and esophageal cancer, and preferably CRC cells.

The invention further relates to peptide-based biomarkers according to the invention, referred to herein as "targets," which can be used in the diagnosis of cancer, preferably CRC. The marker can be overpresentation of the peptide itself, or overexpression of the corresponding gene. Markers may also be used to predict the success rate of treatment, preferably immunotherapy, most preferably immunotherapy targeting the same targets identified by biomarkers. For example, an antibody or soluble TCR can be used to stain tumor sections and detect the presence of the peptide of interest complexing with MHC.

Optionally, the antibody possesses additional effector functions such as immunostimulatory domains or toxins.

The invention also relates to the use of these novel targets in the context of cancer treatment.

Both therapeutic and diagnostic uses for additional cancerous diseases are disclosed in the following more detailed description of the basic expression product (polypeptide) of the peptide according to the invention.

AKAP8 (also referred to as AKAP95) is a scaffold protein that contains a binding domain for the RI / RII subunit of protein kinase A (PKA) and recruits PKA and other signaling molecules to specific intracellular sites. Encodes members of the A-kinase anchor protein family, including. AKAP8 may play a role in chromosome condensation during mitosis by binding to the RIIα subunit of PKA and targeting the PKA and condensin complexes to chromatin (RefSeq, 2002). AKAP8 protein expression is significantly upregulated in rectal and lung cancers and is associated with cell differentiation and histopathological types, suggesting an important role in tumor development and progression. Expression of AKAP8 correlates with expression of cyclin E and cyclin D (Chen et al., 2012; Hu et al., 2013; Qi et al., 2015).

ARHGAP39 (also referred to as Vilse or CrGAP) encodes a Rho GTPase-activating protein involved in Roundaboout (Robo) receptor-mediated repulsive axon guidance and regulation of RAC-dependent cytoskeletal changes (Hu et). al., 2005). ARHGAP39 is often upregulated in bladder cancer cell lines (Matsuda et al., 2011). ARHGAP39 is involved in the directional migration of endothelial cells (Kaur et al., 2008).

AURKB (also referred to as AIM-1) encodes Aurora kinase B, a member of the Aurora kinase subfamily of serine / threonine kinases. AURKB, along with other proteins, regulates chromosomal segregation during mitosis and meiosis through binding to microtubules (RefSeq, 2002). AURKB expression is upregulated in different cancer types, including lung cancer, colorectal cancer, and breast and leukemia, thereby being associated with a poor prognosis. Therefore, the development of OURKB inhibitors for clinical therapy is an interesting area (Hayama et al., 2007; Pohl et al., 2011; Hegyi et al., 2012; Goldenson and Crispino, 2015). Overexpression of AURKB results in phosphorylation of histone H3 and chromosomal instability, a key component of carcinogenesis (Ota et al., 2002; Tatsuka et al., 1998). AURKB activity enhances carcinogenic Ras-mediated cell transformation (Kanda et al., 2005).

C18orf21 (also referred to as XTP13) encodes an abnormal hemoglobin β-chain peptide associated with inhibition of HIV infection (Liu et al., 2011a; Aschauer et al., 1983).

CCNB1 encodes cyclin B1, a regulatory protein involved in mitosis. It has two alternative transcripts, one constitutively expressed and the other predominantly expressed during the G2 / M phase (RefSeq, 2002). CCNB1 encodes cyclin B1, a regulatory protein involved in mitosis. (RefSeq, 2002). CCNB1 is a well-described tumor antigen, and overexpression of CCNB1 has been described for breast cancer, head and neck cancer, prostate cancer, colorectal cancer, lung cancer, and liver cancer (Egloff et al. , 2006). CCNB1 has been shown to be upregulated in a variety of cancer entities, including colorectal cancer, breast cancer, lung cancer, and kidney cancer. Downregulation of CCNB1 results in G2 / M phase cell cycle arrest and inhibition of proliferation and migration (Chang et al., 2013; Sakurai et al., 2014; Fang et al., 2014; Ding et al., 2014). Gene polymorphisms in the CCNB1 gene are associated with breast cancer susceptibility, progression, and survival in Chinese Han Chinese women (Li et al., 2013).

CCNB2 encodes cyclin B2, a member of the cyclin family that plays a role in cell cycle regulation (RefSeq, 2002). CCNB2 is upregulated in colorectal adenocarcinoma (Park et al., 2007). CCNB2 is overexpressed in various human tumors. Strong CCNB2 expression in tumor cells is associated with a poor prognosis in patients with lung adenocarcinoma and invasive breast cancer (Takashima et al., 2014; Albulescu, 2013).

CCT7 encodes the TCP1-containing chaperonin complex (CCT) subunit 7 (η) involved in ATP-dependent folding of various proteins, including actin and tubulin. CCT7 has been found to be part of a protein subnet that significantly distinguishes late-stage human colorectal cancer (Nibbe et al., 2009).

CDC42BPG (also referred to as MRCKγ) encodes CDC42-binding protein kinase γ (DMPK-like), a member of the myotonic dystrophy kinase-related CDC42-binding kinase family (Ng et al., 2004).

CDC6 encodes a protein essential for the initiation of DNA replication (RefSeq, 2002). CDC6 expression is deregulated in different cancer types, including gallbladder cancer, cervical cancer, and prostate cancer (Wu et al., 2009; Wang et al., 2009c; Loveles et al. , 2002; Shu et al., 2012). CDC6 cooperates with c-Myc to promote genetic instability, tumor-like transformation, and attenuation of apoptosis (Chen et al., 2014a). Hypoxia-induced ATR promotes the degradation of CDC6. The initiation of DNA replication is regulated by p53 through Cdc6 protein stability (Dursma and Agami, 2005; Martin et al., 2012).

CENPE is a kinesin-like motor protein that accumulates during the G2 phase of the cell cycle. It is proposed that CENPE, which encodes the centromere protein E, 312 kDa, is one of the motors responsible for mammalian chromosome migration and / or spindle elongation. (RefSeq, 2002). CENPE expression significantly correlates with the grade of glioma and may supplement other parameters for predicting survival time in glioma patients (Bie et al., 2011). CENPE is upregulated in chemotherapy-resistant epithelial ovarian tumors as compared to chemotherapy-sensitive tumors (Ju et al., 2009). CENPE is upregulated in invasive and invasive prolactin pituitary tumors (Wierincx et al., 2007).

CIRH1A (also referred to as Cirhin) encodes a cirrhotic autosomal recessive 1A, a WD40 repeat-containing protein localized in the nucleolus. It causes North American Indian childhood cirrhosis (NAIC) (RefSeq, 2002). CIRH1A can upregulate standard NF-κB factors and may be involved in the regulation of other genes containing NF-κB factors. This suggests that CIRH1A may affect the cancer-related NF-κB pathway (Yu et al., 2009).

CNOT1 encodes an enzymatically relevant subunit of the CCR4-NOT deadenylase complex, an important regulator of translation and mRNA stability (Ito et al., 2011; Bond et al., 2013). A single nucleotide polymorphism (SNP) of the CNOT1 gene was detected in osteosarcoma and acute lymphoblastic leukemia (ALL) (Gutierrez-Camino et al., 2014; Bilbao-Aldaiturriaga et al., 2015). CNOT1 depletion induces mRNA stabilization and activation of ER stress-mediated apoptosis (Ito et al., 2011).

COL12A1 encodes the alpha chain of type XII collagen, a member of the FACIT (fibril-related collagen with interrupted triple helix) collagen family and part of the extracellular matrix (ECM) (RefSeq, 2002). COL12A1 is overexpressed in drug-resistant mutants of ovarian cancer cell lines (Januchowski et al., 2014). In colorectal cancer, COL12A1 is overexpressed in fibrogenic stroma and surrounding cancer-related fibroblasts, as well as in cancer cells that line the infiltrating anterior surface (Karagiannis et al., 2012).

CYP2W1 encodes a member of the cytochrome P450 superfamily of enzymes, a monooxygenase that catalyzes multiple reactions involved in drug metabolism and in the synthesis of cholesterol, steroids, and other lipids (RefSeq, 2002). .. CYP2W1 is overexpressed in a variety of human cancers, including hepatocellular carcinoma, colorectal cancer, and gastric cancer. CYP2W1 overexpression is associated with tumor progression and poor survival (Aung et al., 2006; Gomez et al., 2010; Zhang et al., 2014e). Due to tumor-specific expression, CYP2W1 is an interesting drug target in the treatment of cancer or an enzymatic activator of a prodrug (Karlgren and Ingelman-Sundberg, 2007; Nishida et al., 2010).

ECT2 encodes an epithelial cell transforming protein 2, a guanine nucleotide exchange factor and transforming protein associated with Rho-specific exchange factors and cell cycle regulators (RefSeq, 2002). ECT2 is overexpressed as a result of tumor-specific gene amplification in a variety of human tumors, including lung, ovarian, gastric, and pancreatic cancers. ECT2 is important for cell proliferation, migration, infiltration, and tumorigenicity (Fields and Justilien, 2010; Jin et al., 2014). Protein kinases Cι and ECT2 are activated through MEK / ERK and signal tumor-initiated cell phenotype in ovarian cancer (Wang et al., 2013e). Nuclear ECT2 preferentially binds to Rho GTPase Rac1 and leads to cell transformation through Rac1 activation, while cytoplasmic ECT2 binds to Rho GTPase RhoA and leads to cytokinesis groove formation through RhoA activation (Su). et al., 2011; Huff et al., 2013).

GPR56 encodes an adhesive G protein-coupled receptor G1 (ADGRG1) that regulates cerebral cortex pattern formation. GPR56 specifically binds to transglutaminase 2, a tumor progression inhibitor (RefSeq, 2002). GPR56 inhibits tumorigenesis by suppressing tumor growth and metastasis in melanoma and prostate cancer. Its role in other cancer types appears to be complex, presumably due to the various ability of different splicing variants of GPR56 to activate transcription factors such as c-myc and p53 response elements (Kim et al). ., 2010b; Xu et al., 2010; Yang and Xu, 2012). GPR56 inhibits VEGF production from melanoma cells and inhibits their angiogenesis and proliferation through signaling pathways involving the protein kinase Cα (Yang et al., 2011a).

HS6ST2 encodes a member of the heparin sulfate (HS) sulfotransferase gene family. HS6ST2 catalyzes the transfer of sulfate to HS (RefSeq, 2002). HS6ST2 is overexpressed in different cancer types, including thyroid cancer, colorectal cancer, and ovarian cancer, and is associated with migration, infiltration, and poor prognosis (Backen et al., 2007; Hatabe et al). ., 2013; Di et al., 2014). TGF-β promotes cancer metastasis by stimulating HS6ST2 and IL-11 production (Pollari et al., 2012). HS6ST2 is a regulator of angiogenesis in response to EGF, FGF2, and VEGF signaling pathways (Ferreras et al., 2012; Cole et al., 2014).

IER3 (also referred to as IEX-1) encodes the earliest response 3 which has the function of protecting cells from Fas or tumor necrosis factor α-induced apoptosis (RefSeq, 2002). Deregulation of IER3 expression in ovarian, pancreatic, blood, breast, colorectal, lymphoma, and myeloma is associated with poor or better prognosis, depending on the type and stage of progression of the tumor. And make the protein a valuable biomarker, either alone or in combination with other genes (Wu et al., 2013). IER3 gene expression plays an important role in regulating apoptosis and cell proliferation through positive and negative modalities. Overexpression of IER3 sensitizes some cells to apoptosis and accelerates cell cycle progression, whereas inhibition of IER3 expression slows cell cycle progression, whereas inhibition of IER3 expression is both apoptosis and cell cycle progression. (Zhang et al., 2011a). IER3 interferes with specific signaling pathways, especially NF-κB, MAPK / ERK, and PI3K / Akt. The mouse model also revealed the involvement of IER3 expression in immune function (Alt and Schafer, 2011; Wu, 2003).

ITPR3 encodes a receptor for inositol 1,4,5-trisphosphate, which contains a C-terminal calcium channel and an N-terminal ligand binding site. ITPR3 plays a role in the exocrine glands underlying energy metabolism and proliferation (RefSeq, 2002). ITPR3 is overexpressed in several cancer types, including colorectal cancer, gastric cancer, and breast cancer, and is directly associated with cancer progression and tumor invasiveness (Shibao et al., 2010; Mound et al.). ., 2013; Sakakura et al., 2003). Akt can protect cells from apoptosis in an ITPR3-dependent manner by reducing Ca2 + release from the endoplasmic reticulum (Marchi et al., 2012).

KCNN4 (also referred to as KCa3.1 or hIKCa1) encodes part of the heterotetramer voltage independent potassium channel activated by intracellular calcium. Activation of this channel is followed by membrane hyperpolarization, which promotes calcium influx (RefSeq, 2002). KCNN4 is upregulated in several cancers, including breast, lung, and prostate cancers and is associated with cell proliferation and tumor proliferation (Chou et al., 2008; Lallet-Daher et al., 2009; Haren et al., 2010; Bulk et al., 2015). Inhibition of KCNN4 regulates reactive oxygen species (ROS) levels to promote p53 activation, which suppresses cell proliferation and migration and leads to apoptosis (Liu et al., 2015b).

KIRREL (also referred to as NEPH1) encodes a member of the nephrin-like protein family, of which its members interact with the cytoplasmic domain of podosine (RefSeq, 2002).

KLK10 (also referred to as NES1) encodes a member of the kallikrein subfamily of serine proteases, which plays a role in carcinogenesis and has potential as a biomarker (RefSeq, 2002). KLK10 is upregulated in colon, ovarian, and gastric cancers, but downregulated in breast, lung, and prostate cancers (Yousef et al., 2005; Feng et al., 2006; Zhang et. al., 2010; Li et al., 2001). Epigenetic silencing of KLK10 is maintained by TGFbeta / Smad signaling, while KLK10 upregulation is facilitated by activation of Ras / MEK / ERK and PI3K / Akt signaling (Pariouras and Diamandis, 2008). Papageorgis et al., 2010).

LIG1 is a DNA repair gene involved in the nucleotide excision repair (NER) and base excision repair (BER) pathways. LIG1 single nucleotide polymorphisms are associated with lung cancer, endometrial cancer, and glioma risk (Doherty et al., 2011; Lee et al., 2008b; Liu et al., 2009).

LSG1 encodes the large 60S subunit nucleus xxportGTPase 1. The protein is required for cell survival and may be localized to the endoplasmic reticulum, nucleus, and cytoplasm (RefSeq, 2002).

LSM14B (also referred to as RAP55B) is involved in RNA metabolism, regulation of mitotic G2 / M phase, translational repression, uptake into mRNP particles, P-bodies formation, and stress granule localization, LSM (Sm). M) Codes members of the domain family (Marnef et al., 2009; Albrecht and Lengauer, 2004).

MAGED2 encodes melanoma antigen families D2, which are members of the newly defined MAGE-D cluster in Xp11.2, a hotspot for X-chain mental retardation. MAGED2 is ubiquitously expressed at high expression levels in specific brain regions and testicular stroma. MAGED2 is a potential negative regulator of wild-type p53 activity (Langnaese et al., 2001; Papageorgio et al., 2007). Overexpression of MAGED2 is associated with melanoma, breast cancer, and colon cancer (Li et al., 2004; Strekalova et al., 2015).

MAGEF1 encodes a member of the melanoma antigen (MAGE) superfamily, which is ubiquitously expressed, including microsatellite repeats, suggesting a role in normal cell physiology (Stone et al., 2001). Flavopyridol induces inhibition of human tumor cell proliferation and downregulation of MAGEF1 in different human tumor cell lines (Lu et al., 2004). MAGEF1 is significantly overexpressed in colorectal cancer tissues (Chung et al., 2010).

MDH1 encodes malate dehydrogenase, an enzyme that catalyzes the reversible oxidation of malate to oxaloacetate by utilizing the NAD / NADH cofactor system in the citric acid cycle. MDH1 may play a central role in the malate-aspartate shuttle, which is localized in the cytoplasm and functions in the metabolic coordination between the cytosol and mitochondria (RefSeq, 2002). In glioblastoma, MDH1 is the target of several deregulated microRNAs whose expression is suppressed. Along with known tumor suppressors or carcinogens, MDH1 can help distinguish between low-grade and high-grade gliomas (Lages et al., 2011; Kounelakis et al., 2013). MDH1 is overexpressed in null cell pituitary adenomas and thyroid oncocytomas (Hu et al., 2007; Baris et al., 2004).

MYO10 encodes a member of the myosin superfamily, which corresponds to atypical myosin. It functions as an actin-based molecular motor and plays a role in the integration of F-actin with the microtubule cytoskeleton during meiosis (RefSeq, 2002). MYO10 is overexpressed in several cancer entities, including breast and lung cancer, and is associated with metastasis, cell migration, and invasive phenotype (Cao et al., 2014; Sun et al., 2015b; Curson). and Chainey, 2015). Mutant p53 promotes (Mutant p53 promotes) NCAPG encodes the non-SMC condensed I complex subunit G, which is responsible for the condensation and stabilization of chromosomes during mitosis and meiosis (RefSeq, 2002). .. NCAPG is downregulated in patients with multiple myeloma, acute myeloid leukemia, and leukemia cells derived from blood or myeloma cells (Cohen et al., 2014). NCAPG may be a multidrug resistance gene in colorectal cancer (Li et al., 2012). NCAPG is highly upregulated in the chromophobe subtypes of human cell carcinoma, but not in conventional human renal cell carcinoma (Kim et al., 2010a). Upregulation of NCAPG is associated with melanoma progression (Ryu et al., 2007). NCAPG is associated with uveal melanoma (Van Ginkel et al., 1998). NCAPG exhibits variable expression in different tumor cells (Jager et al., 2000).

NDRG3 encodes a member of the N-myc downstream regulatory gene, which is highly expressed in the testis, prostate, and ovary and may play a role in spermatogenesis (Zhao et al., 2001). NDRG3 may function as a tumor suppressor gene for different cancer types, including bladder cancer (Yang et al., 2013; Tsui et al., 2015). NDRG3 acts as a tumor promoter in prostate cancer, with upregulated expression leading to increased growth rate, higher migration, and induction of angiogenic chemokines. Upregulation of NDRG3 is associated with a malignant phenotype in hepatocellular carcinoma cells (Wang et al., 2009b; Fan et al., 2011).

NOL11 encodes the nucleolar protein 11 required for optimal rDNA transcription in ribosome biogenesis (Freed et al., 2012; Griffin et al., 2015). Nol11 is an interacting agent of the breast and ovarian tumor suppressor BRCA1 (Hill et al., 2014).

PLAGL2 is a zinc finger protein that encodes a member of the pleomorphic adenoma gene (PLAG) family and recognizes DNA and / or RNA (Kas et al., 1998). PLAGL2 functions as a proto-oncogene in a variety of cancers, including leukemia, glioma, colorectal cancer, and lung adenocarcinoma. There is also evidence that PLAGL2 can act as a tumor suppressor by initiating cell cycle arrest and apoptosis (Yang et al., 2011b; Hanks and Gauss, 2012; Liu et al., 2014a). PLAGL2 prevents proteasome degradation of the E3 ubiquitin ligase Pirh2, which regulates the stability of p53. PLAGL2 expression also increases p73 levels and upregulates p73 target genes such as p73 and Bax (Zheng et al., 2007; Hanks and Gauss, 2012; Landrette et al., 2005).

PTCD2 encodes a pentatrichopeptide repeat domain protein 2 that may be involved in the processing of RNA transcripts, including cytochrome b derived from mitochondrial DNA. Dysfunction of this protein plays a possible role in the pathogenesis of heart failure (Xu et al., 2008).

RAD54 encodes a protein belonging to the DEAD-like helicase superfamily. It shares similarities with Saccharomyces cerevisiae RAD54 and RDH54, both of which are involved in DNA homologous recombination and repair. This protein binds to double-stranded DNA and exhibits ATPase activity in the presence of DNA. This gene is highly expressed in the testis and spleen, suggesting an active role in meiosis and mitosis recombination (RefSeq, 2002). Homozygous variants of RAD54B were observed in primary lymphoma and colon cancer (Hiramoto et al., 1999). RAD54B suppresses the genomic destabilizing effect of direct binding of RAD51 to dsDNA in human tumor cells (Mason et al., 2015).

RNASEH2A encodes a component of the heterotrimer type II ribonuclease H enzyme and is a major source of its activity. RNASEH2A is an endonuclease and is predicted to remove Okazaki fragment RNA primers during lagging strand DNA synthesis (RefSeq, 2002). RNASEH2A is upregulated in transformed mesenchymal stem cells and is overexpressed in many cancer cells, including invasive prostate cancer. Knockdown of RNASEH2A inhibits scaffold-independent growth but does not alter cancer cell growth (Flangan et al., 2009; Williams et al., 2014).

RRM1 encodes the ribonucleotide reductase M1, which is an essential enzyme for the production of deoxyribonucleotides prior to DNA synthesis in the S phase of dividing cells. It is one of several genes located in the imprinting gene domain of 11p15.5, an important tumor suppressor gene region (RefSeq, 2002). RRM1 is involved in the regulation of cell proliferation, cell migration, tumorigenesis, and metastatic progression. Studies involving a large number of patients with different cancer types such as lung cancer, pancreatic cancer, breast cancer, and gastric cancer establish the prognostic or predictive value of RRM1 (Carvalho et al., 2009; Jordan et al. , 2011; Wang et al., 2013d). The nucleoside analog gemcitabine, a common chemotherapeutic agent in the treatment of cancer, targets RRM1 (Jordhem and Dumontet, 2013).

SERPINB5 (also called maspin) encodes the serpin peptidase inhibitor Clade B member 5, which is characterized as a class II tumor suppressor based on its ability to promote apoptosis and inhibit cell infiltration and angiogenesis (Bailey). et al., 2006). SERPINB5 is a useful molecular marker for diagnosis and a predictor of prognosis for many cancer types, including breast, lung, head and neck cancer, oral cancer, and prostate cancer (Marioni et). al., 2009; Lonardo et al., 2010; Sager et al., 1996; Sheng, 2004). SERPINB5 plays the role of an endogenous regulator of HDAC1 activity and interacts with the p53 tumor suppressor pathway (Mass et al., 2000; Kaplun et al., 2012).

SEZ6L2 encodes a seizure-related protein localized on the cell surface and abundant in pancreatic β-cells (RefSeq, 2002; Stutzer et al., 2013). Expression of SEZ6L2 is upregulated in lung cancer, with higher expression levels associated with shorter survival (Ishikawa et al., 2006).

SMARCA4 (also referred to as BRG1) is part of the large ATP-dependent chromatin remodeling complex SWI / SNF, which usually encodes a member of the SWI / SNF family of helicase and ATPase-containing proteins. Is required for transcriptional activation of genes suppressed by chromatin (RefSeq, 2002). SMARCA4 acts as a tumor suppressor and is downregulated through mutations in different cancer entities, including breast, lung, and colon cancers. Low SMARCA4 levels are associated with tumor progression such as mutation and infiltration (Medina and Sanchez-Cespedes, 2008; Bai et al., 2013b; Reisman et al., 2003; Wang et al., 2016). SMARCA4 is associated with several tumor suppressors and important tumor-related proteins such as p53, p16INK4a, hTERT, and Akt (Medina and Sanchez-Cepsedes, 2008; Becker et al., 2009; Naidu et al. , 2009; Liu et al., 2014b; Wu et al., 2014a).

SMC2 (also referred to as CAP-E or SMC2L1) encodes a member of the structural maintenance of the chromosomal family important for mitotic chromosome condensation and DNA repair (RefSeq, 2002). The SMC2 gene is modified by frameshift mutations, and loss of expression in gastric and colorectal cancers with microsatellite instability suggests that SMC2 may be involved in the pathogenesis of tumors (Je et al.,. 2014). Modifications of the SMC2 gene may play a role in genomic instability, promoting the accumulation of other modifications in empyema-related lymphoma (Ham et al., 2007).

SVIL encodes supervillin, a dichotomous protein with distinct amino-terminal and carboxy-terminal domains that appears to aid in myosin II construction during cell diffusion and focal adhesions (RefSeq, 2002). SVIL is significantly downregulated in prostate cancer tissue, primarily through promoter methylation (Vanaja et al., 2006). SVIL regulates cell survival through regulation of p53 levels. SVIL expression is required for crosstalk between survival signaling and cellular motor pathways (Fang and Luna, 2013).

TMEM222 encodes a transmembrane protein 222 located on chromosome 1p36.11. (RefSeq, 2002).

ZNF679 encodes a zinc finger protein containing a KRAB (Kruppel-related box) domain that functions as a transcription factor. The promoter region of ZNF679 is bound by co-inhibitors KAP1 and H3me3K9 (histone 3 trimethylation of lysine 9) (O'Gene et al., 2007).

Stimulation of the immune response depends on the presence of antigens recognized as foreign by the host immune system. The discovery of the presence of tumor-related antigens has increased the likelihood of using the host's immune system to intervene in tumor growth. Various mechanisms that utilize both the humoral and cellular arms of the immune system are currently being explored for cancer immunotherapy.

Certain components of the cell-mediated immune response can specifically recognize and destroy tumor cells. Isolation of T cells from tumor-infiltrating cell populations or from peripheral blood suggests that such cells play an important role in the innate immune defense against cancer. In particular, CD8-positive T that recognizes class I molecules of major histocompatibility complex (MHC) -carrying peptides, usually derived from proteins located within the cytosol or defective ribosome products (DRIPS), usually with 8-10 amino acid residues. Cells play an important role in this response. Human MHC molecules are also referred to as human leukocyte antigen (HLA).

In the usage herein, all terms are defined as described below, unless otherwise stated.

The term "T cell response" means the specific proliferation and activation of effector function induced by peptides in vitro or in vivo. In MHC class I constrained cytotoxic T cells, the effector function is peptide-pulsed, peptide precursor-pulsed or native peptide-presenting target cell lysis; preferably peptide-induced interferon-γ, TNF-α, Alternatively, it may be the secretion of a cytokine that is IL-2; preferably a peptide-induced secretion of an effector molecule that is a granzyme or perforin; or degranulation.

The term "peptide" is used herein to name a series of amino acid residues that are typically linked together by peptide bonds between the α-amino and carbonyl groups of adjacent amino acids. The peptide is preferably 9 amino acids long, but can be as short as 8 amino acids and as long as 10, 11, 12, 13, or 14 or more, in the case of MHC class II peptides (peptides of the invention). (Extended variants of), they can be 15, 16, 17, 18, 19 or longer than 20 amino acids in length.

Further, the term "peptide" is intended to include salts of a series of amino acid residues that are typically linked to each other by peptide bonds between the α-amino and carbonyl groups of adjacent amino acids. Preferably, the salt is a pharmaceutically acceptable salt of the peptide, for example a chloride salt or an acetate salt (trifluoroacetate). It should be noted that since peptides are not salts in vivo, the salts of peptides according to the invention are substantially different in their in vivo state from peptides.

The term "peptide" shall also include "oligopeptide". The term "oligopeptide" is used herein to name a series of amino acid residues that are typically linked together by peptide bonds between the α-amino and carbonyl groups of adjacent amino acids. .. The length of the oligopeptide is not important to the invention as long as the correct epitope or epitope is retained therein. Oligopeptides are typically less than about 30 amino acid residue lengths and more than about 15 amino acid lengths.

The term "polypeptide" typically refers to a series of amino acid residues that are linked together by peptide bonds between the α-amino and carbonyl groups of adjacent amino acids. The length of the polypeptide is not important to the invention as long as the correct epitope is retained. In contrast to the term peptide or oligopeptide, the term polypeptide is intended to refer to a molecule containing more than about 30 amino acid residues.

A peptide, oligopeptide, protein or polynucleotide encoding such a molecule is "immunogenic" if it can elicit an immune response (hence the "immunogen" in the present invention). In the present invention, immunogenicity is more specifically defined as the ability to induce a T cell response. Therefore, an "immunogen" is a molecule capable of inducing an immune response, and in the present invention, a molecule capable of inducing a T cell response. In another aspect, the immunogen can be a peptide, a complex of peptide and MHC, an oligopeptide, and / or a protein used to generate a specific antibody or TCR against it.

Class IT cell "epitope" requires a short peptide bound to a class I MHC receptor, forming a tricomponent complex (MHC class Iα chain, β-2-microglobulin, and peptide), which is Can be recognized by T cells carrying compatible T cell receptors that bind to the MHC / peptide complex with appropriate affinity. Peptides that bind to MHC class I molecules are typically 8-14 amino acids long, most typically 9 amino acids long.

In humans, there are three different loci, HLA-A, HLA-B, and HLA-C, which encode MHC class I molecules (human MHC molecules also also referred to as human leukocyte antigens (HLA)). HLA-A ^* 01, HLA-A ^* 02, and HLA-B ^* 07 are examples of different MHC class I alleles that can be expressed from these loci.

Table 5: HLA-A ^* 02 and HLA-A ^* 24 expression frequency F, and the most frequent HLA-DR serotypes. Frequency is a haplotype frequency Gf within the American population adapted from Mori et al. (Mori et al., 1997) using the Hardy-Weinberg formula, F = 1- (1-Gf) ². Estimated from. Due to linkage disequilibrium, combinations of A ^* 02 or A ^* 24 with specific HLA-DR alleles may be more abundant or less frequent than expected from their single frequency. .. See Chanock et al. (Chanock et al., 2004) for more information.

The peptides of the invention preferably bind to ^{A *} 02 when included in the vaccines of the invention described herein. The vaccine may also contain a pan-bound MHC class II peptide. Therefore, while the vaccines of the invention can be used to ^{treat cancer in A *} 02 positive patients, it is not necessary to select the MHC class II allotype because of the pan-binding properties of these peptides.

When the A ^* 02 peptide of the ^{invention is combined with a peptide that binds to another allele, such as A *} 24, of any patient population compared to treatment with any single MHC class I allele. A higher percentage can be treated. In the majority population, less than 50% of patients can be treated with any single allele, while ^{vaccines containing HLA-A *} 24 and HLA-A ^* 02 epitopes are any reasonable mother. At least 60% of patients in the population can be treated. Specifically, in various regions, the following percentages of patients are positive for at least one of these alleles: US 61%, Western Europe 62%, China 75%, South Korea 77%, Japan 86% ( Calculated from www.allelefrequies.net).

In a preferred embodiment, the term "nucleotide sequence" refers to a heteropolymer of deoxyribonucleotides.

Nucleotide sequences encoding a particular peptide, oligopeptide, or polypeptide may be of natural origin, or they may be synthetically constructed. In general, the DNA fragments encoding the peptides, polypeptides, and proteins of the invention are constructed from cDNA fragments and short oligonucleotide linkers, or from a series of oligonucleotides, and are regulators derived from microorganisms or viral operons. Provided are synthetic genes that can be expressed in recombinant transcription units, comprising:

As used herein, the term "nucleotide coding (or encoding) a peptide" is used in a sequence that is compatible with a biological system in which the sequence is expressed, for example, by a dendritic cell or another cell line useful for the production of the TCR. Refers to a nucleotide sequence that encodes a peptide, including certain artificial (artificial) start and stop codons.

As used herein, reference to nucleic acid sequences includes both single-stranded and double-stranded nucleic acids. Thus, for example, a particular sequence may be a single-stranded DNA of such a sequence, a double-stranded DNA of such a sequence and its complement (double-stranded DNA), unless the context clearly suggests a different meaning. , And the complement of such an array.

The term "coding region" refers to a portion of a gene that naturally or normally encodes a gene expression product within its natural genomic environment, i.e., a region that encodes the naturally occurring product of a gene in vivo.

The coding region can be derived from a non-mutated (“normal”), mutated or modified gene, or a DNA sequence or gene fully synthesized in the laboratory using methods well known to those of skill in the art of DNA synthesis techniques. Can even be derived from.

The term "expression product" means a polypeptide or protein that is a natural translation of any nucleic acid sequence that encodes a gene and thus an equivalent due to genetic code degeneration and thus encodes the same amino acid.

When referring to a coding sequence, the term "fragment" is a DNA whose expression product contains a less than complete coding region that retains essentially the same biological function or activity as the expression product of the complete coding region. Means the part of.

The term "DNA fragment" refers to a DNA polymer in the form of separate fragments or as a component of a larger DNA construct, which is substantially pure, i.e. free of contaminating endogenous substances. The fragment and its constituent nucleotide sequences are derived from the DNA isolated at least once in an amount or concentration that can be identified, manipulated, and recovered, eg, by standard biochemical methods using cloning vectors. Such fragments are provided in the form of a reading frame, typically uninterrupted by internal untranslated sequences or introns present within eukaryotic genes. The untranslated DNA sequence may be located downstream of the reading frame, as it does not interfere with manipulation or expression of the coding region.

The term "primer" means a short nucleic acid sequence that can be paired with a single strand of DNA and provides a free 3'-OH terminal where the DNA polymerase initiates deoxyribonucleotide strand synthesis.

The term "promoter" means a region of DNA involved in RNA polymerase binding to initiate transcription.

The term "isolated" means that the substance is taken from its original environment (eg, the natural environment if it is of natural origin). For example, a natural polynucleotide or polypeptide present in a living animal has not been isolated, but the same polynucleotide or polypeptide isolated from some or all of the substances coexisting in the natural system has been isolated. There is. Such polynucleotides can be part of a vector and / or such polynucleotides or polypeptides can be part of a composition, such a vector or composition being one of its natural environment. It is still isolated in the sense that it is not a part.

The polynucleotides disclosed by the present invention, as well as recombinant or immunogenic polypeptides, may be in "purified" form. The term "purification" does not have to be completely pure; rather, it is intended to be a relative definition and highly purified preparations as these terms are understood by those of skill in the art. , Or only partially purified preparations. For example, individual clones isolated from a cDNA library are electrophoretically and uniformly purified by conventional methods. Purification of starting materials or natural materials of at least 1 digit, preferably 2 or 3 digits, more preferably 4 or 5 digits is explicitly considered. Further, by weight, the claimed polypeptide is expressly included, preferably 99.999%, or at least 99.99% or 99.9%; even more preferably 99% or higher purity.

Nucleic acid and polypeptide expression products disclosed by the present invention, as well as expression vectors containing such nucleic acids and / or such polypeptides, may be in "enriched form". As used herein, the term "enrichment" means that the concentration of a substance is (eg) at least about 2, 5, 10, 100, or 1000 times its natural concentration, advantageously by weight. It is 0.01% by weight, preferably at least about 0.1% by weight. Enriched preparations of about 0.5%, 1%, 5%, 10%, and 20% by weight are also considered. The sequences, constructs, vectors, clones, and other substances that make up the invention can advantageously be enriched or isolated forms. The term "active fragment" is usually administered alone or optionally with an appropriate adjuvant, or in a vector to animals such as rabbits or mice and also humans and other mammals. It means a fragment of a peptide, polypeptide or nucleic acid sequence that then yields an immune response (ie, has immunogenicity), such an immune response is a form that stimulates a T cell response in a recipient animal such as a human. take. Alternatively, "active fragments" may also be used to induce in vitro T cell responses.

As used herein, the terms "partial," "fragment," and "fragment," as used in the context of a polypeptide, refer to a sequence of contiguous residues, such as amino acid residues, the sequence thereof. Form a subset of a larger array. For example, if the polypeptide is treated with either trypsin or a common endopeptidase such as chymotrypsin, the oligopeptide obtained from such treatment will correspond to a portion, fragment or fragment of the starting polypeptide. Let's go. When used with respect to polynucleotides, these terms refer to the product of processing the polynucleotide with any endonuclease.

According to the invention, when referring to a sequence, the terms "identity percentage" or "percently identical" are the sequence being compared ("comparative sequence") and the sequence described or claimed ("reference sequence"). It means that after alignment with), the sequence is compared to the sequence claimed or described. The identity percentage is then determined according to the following equation:
Identity Percentage = 100 [1- (C / R)]
In the equation, C is the number of differences between the reference sequence and the comparison sequence over the alignment length between the reference sequence and the sequence to be compared.
With (i) each base or amino acid in the reference sequence that does not have a corresponding aligned base or amino acid in the comparison sequence, and (ii) each gap in the reference sequence, and (iii) the aligned base or amino acid in the comparison sequence. Different, each aligned base or amino acid in the reference sequence constitutes a difference,
(Iiii) Alignment must start at position 1 of the matching sequence;
R is the number of bases or amino acids in the reference sequence over the alignment length with the comparison sequence, and any gaps that occur in the reference sequence are also counted as bases or amino acids.

If there is an alignment between the comparison sequence and the reference sequence for which the identity percentage is calculated as above, which is approximately equal to or greater than the particular minimum identity percentage, then the above The comparison sequence has a particular minimum identity percentage with the reference sequence, even if there are alignments where the identity percentage so calculated is less than a particular identity percentage.

Thus, as described above, the invention is a variant thereof that is 88% homologous to SEQ ID NO: 1 to SEQ ID NO: 191 or SEQ ID NO: 1 to SEQ ID NO: 191 or a variant thereof that causes T cells to cross-react with the peptide. Provided is a peptide comprising a sequence selected from the group consisting of. The peptides of the invention have the ability to bind human major histocompatibility complex (MHC) class I molecules or extended versions of said peptides to class II.

In the present invention, the term "homologous" refers to the degree of identity between two amino acid sequences, i.e., a sequence of a peptide or polypeptide sequence (see Identity Percentage above). The aforementioned "homology" is determined by comparing two sequences aligned under optimal conditions across the sequences being compared. Such sequence homology can be calculated, for example, by creating an alignment using the ClustalW algorithm. Generally available sequence analysis software, more specifically Vector NTI, GENETYX or other tools, are provided by public databases.

One of skill in the art will be able to assess whether T cells induced by variants of a particular peptide can cross-react with the peptide itself (Appay et al., 2006; Colombetti et al., 2006; Fong et. al., 2001; Zaremba et al., 1997).

Due to the "variant" of a given amino acid sequence, we still have an HLA molecule as well as a peptide consisting of a given amino acid sequence consisting of SEQ ID NOs: 1 to SEQ ID NO: 191. Changes in the side chains of one or two residues of an amino acid, for example (eg, by substituting them with a side chain of another natural amino acid residue or another side chain) so that they can bind. Means. For example, the peptide may be modified to at least maintain, without improving its ability to interact and bind to the binding groove of a suitable MHC molecule such as ^{HLA-A * 02 or -DR.} Thus it at least maintains the ability of activated CTLs to bind to TCR without improvement.

These T cells can subsequently cross-react with the cells to kill cells expressing the polypeptide containing the native amino acid sequence of the cognate peptide defined in aspects of the invention. As can be derived from academic literature and databases (Rammensee et al., 1999; Godkin et al., 1997), the specific position of the HLA-binding peptide is typically an anchor residue and constitutes a binding groove. It forms a core sequence that is compatible with the HLA receptor binding motif defined by the polarity, electrophysical, hydrophobic, and spatial properties of the polypeptide chain. Therefore, one of ordinary skill in the art can modify the amino acid sequences set forth in SEQ ID NOs: 1 to SEQ ID NO: 191 by preserving known anchor residues, and the ability of such variants to bind to MHC class I or II molecules. You will be able to determine if you want to keep it. Variants of the invention maintain the ability of activated T cells to bind to TCR, which subsequently expresses polypeptides containing the natural amino acid sequences of cognate peptides as defined in aspects of the invention. Can cross-react with and kill it.

The original (unmodified) peptide disclosed herein can be modified by the substitution of one or more residues at different, perhaps selective sites within the peptide chain, unless otherwise specified. Preferably these substitutions are located at the end of the amino acid chain. Such substitutions may be conservative in nature, where amino acids are replaced by amino acids with similar structures and characteristics, for example, a hydrophobic amino acid is replaced by another hydrophobic amino acid. Even more conservative substitutions are substitutions of amino acids of the same or similar size and chemistry, such as substitutions of leucine with isoleucine. In studies of sequence diversity of naturally occurring homologous protein families, certain amino acid substitutions are often more tolerated than others, and they are the size, charge, polarity, and hydrophobicity between the original amino acid and its substitutions. It often correlates with gender homology, which is the basis of the definition of "conservative substitution".

Conservative substitutions are defined herein as exchanges within one of five groups: Group 1-small aliphatic, non-polar or slightly polar residues (Ala, Ser, Thr, Pro). , Gly); Group 2-Polar negatively charged residues and their amides (Asp, Asn, Glu, Gln); Group 3-Polar positively charged residues (His, Arg, Lys); Group 4 -Large aliphatic non-polar residues (Met, Leu, Ile, Val, Cys); and Group 5-Large aromatic residues (Phe, Tyr, Trp).

Less conservative substitutions may involve substitutions with other amino acids with similar characteristics but somewhat different sizes, such as substitutions with isoleucine residues of alanine. Highly non-conservative substitutions may involve substitutions of polar amino acids or basic amino acids with acidic amino acids. However, such "extreme" substitutions are potentially ineffective, as chemical effects are not completely predictable and extreme substitutions can result in accidental effects that cannot be predicted by simple chemical principles. Cannot be rejected as.

Of course, structures other than the usual L-amino acids may be involved in such substitutions. Thus, the D-amino acid may replace the L-amino acid commonly found in the antigenic peptides of the invention and is still included in the disclosure herein. In addition, non-standard amino acids (ie, other than common intrinsically disordered amino acids) may also be used for substitution purposes to produce immunogen and immunogenic polypeptides according to the invention.

If substitutions at two or more positions are found to result in peptides with substantially equivalent or greater antigenic activity as defined below, combinations of these substitutions are tested and the substitutions are made. It is determined whether the combination has an additive or synergistic effect on the antigenicity of the peptide. At most, no more than four positions within the peptide are simultaneously substituted.

Peptides consisting essentially of amino acid sequences as shown herein have substantially altered ability to bind to human major histocompatibility complex (MHC) class I or II molecules when compared to unmodified peptides. It may have one or two non-anchor amino acids that are exchanged without being adversely affected (see below for anchor motifs). In another embodiment, in a peptide essentially consisting of an amino acid sequence as shown herein, the ability to bind to a human major histocompatibility complex (MHC) class I or II molecule is compared to an unmodified peptide. Thus, one or two amino acids can be exchanged with their conservative exchange partner (see below) without substantial change or adverse effect.

Amino acid residues that do not substantially contribute to interaction with the T cell receptor, with other amino acids whose integration does not substantially affect T cell reactivity and do not preclude binding to the associated MHC. Can be modified by substitution. Thus, with the exception of the proviso given, the peptides of the invention are any peptides comprising the amino acid sequences or portions or variants thereof as given (the inventors of the invention are oligopeptides or polypeptides in their terminology). Includes).

Table 6: Peptide variants and motifs set forth in SEQ ID NOs: 7, 9, 31, 192, 212, and 142.

Longer (stretched) peptides may also be suitable. MHC class I epitopes are usually 8-11 amino acids long, but can be made by peptide processing from longer peptides or proteins containing the actual epitope. Residues located on the side of the actual epitope are preferably residues that do not substantially affect the proteolytic cleavage required to expose the actual epitope during processing.

The peptides of the invention can be extended with up to 4 amino acids, i.e. any combination between 4: 0 and 0: 4, with 1, 2, 3 or 4 amino acids added to either end. obtain. The combinations of elongation according to the present invention are shown in Table 7.

Table 7: Combinations of peptide elongations of the invention

The amino acid for extension / extension can be a peptide of the original protein sequence or any other amino acid. Elongation can be utilized to increase the stability or solubility of the peptide.

Thus, the epitopes of the invention may be identical to naturally occurring tumor-related or tumor-specific epitopes, or as long as they have substantially the same antigenic activity, no more than four residues are reference peptides. It may contain an epitope different from that of.

In an alternative embodiment, the peptide is unilaterally or bilaterally extended to a total length of more than 4 amino acids, preferably up to 30 amino acids. This may result in an MHC class II binding peptide. Binding to MHC class II can be tested by methods known in the art.

Accordingly, the present invention provides peptides and variants of MHC class I epitopes, the peptide or variant being 8-100, preferably 8-30, most preferably 8-14, ie 8, 9, 10, 11. , 12, 13, 14 amino acids in full length and for extended class II binding peptides, the length can also be 15, 16, 17, 18, 19, 20, 21 or 22 amino acids.

Of course, the peptides or variants according to the invention have the ability to bind to molecules of human major histocompatibility complex (MHC) class I or II. Binding of peptides or variants to MHC complexes may be tested by methods known in the art.

Preferably, when testing peptide-specific T cells according to the invention for a substituted peptide, the peptide concentration at which the substituted peptide achieves half the maximum lysis increase relative to the background is about 1 mM or less, preferably about 1 μM or less. It is more preferably about 1 nM or less, even more preferably about 100 pM or less, and most preferably about 10 pM or less. It is also preferred that the substituted peptide be recognized by T cells from two or more, at least two, more preferably three individuals.

In a particularly preferred embodiment of the invention, the peptide comprises, or consists essentially of, the amino acid sequence set forth in SEQ ID NO: 1 to SEQ ID NO: 191.

"In essence" means that the peptide according to the invention comprises a portion of the peptide that functions as an epitope of an MHC molecular epitope in addition to the sequence set forth in any of SEQ ID NOs: 1 to SEQ ID NO: 191 or a variant thereof. It is meant to contain a set of additional sequences of amino acids located at the N- and / or C-terminus that do not necessarily constitute.

Nonetheless, these sequences of sequences may be important to provide efficient cell introduction of the peptides according to the invention. In one embodiment of the invention, the peptide comprises, for example, 80 N-terminal amino acids of the HLA-DR antigen-related invariant chain (p33, hereinafter "Ii") derived from NCBI, GenBank Accession No. X00497. , Is part of the fusion protein. In other fusions, the peptides of the invention are fused to an antibody as described herein, or a functional portion thereof, particularly to the sequence of the antibody, such that it is specifically targeted by the antibody. It may or may be fused, for example, to or into an antibody specific for dendritic cells as described herein.

In addition, peptides or variants may be further modified to improve stability and / or binding to MHC molecules in order to elicit a stronger immune response. Such methods of optimizing peptide sequences are well known in the art and include, for example, the introduction of reverse peptide or non-peptide bonds.

In the reverse peptide bond, the amino acid residues are not linked by the peptide (-CO-NH-) bond, and the peptide bond is reversed. Such retro-inversopeptide mimetics are known in the art, such as those described in Meziere et al (1997) (Meziere et al., 1997), which is incorporated herein by reference. It may be manufactured using the method of. This approach involves the production of pseudopeptides that contain changes that involve the backbone rather than the direction of the side chains. Meziere et al. (Meziere et al., 1997) show that these pseudopeptides are useful for MHC binding and T-helper cell responses. Retroinverse peptides containing NH-CO bonds instead of CO-NH peptide bonds are much more resistant to proteolysis.

Non-peptide bond, for _{example, -CH 2 -NH, -CH 2 S} -, - CH 2 CH 2 -, - CH = CH -, - COCH 2 -, - CH (OH) CH 2 -, and -CH ₂ SO-. US Pat. No. 4,897,445 involves polypeptides synthesized by standard procedures and non-peptide bonds synthesized by reacting aminoaldehydes with amino acids in the presence of _{NaCNBH 3.} A method for solid-phase synthesis of a non-peptide bond (-CH _{2-NH) in a chain is provided.}

Peptides comprising the above sequences are synthesized with additional chemical groups present at their amino and / or carboxy ends to enhance the stability, bioavailability, and / or affinity of the peptides. You may. For example, a hydrophobic group such as carbobenzoxyl, dansyl, or t-butyloxycarbonyl group may be added to the amino terminus of the peptide. Similarly, an acetyl group or a 9-fluorenylmethoxy-carbonyl group may be located at the amino terminus of the peptide. In addition, a hydrophobic group, t-butyloxycarbonyl, or amide group may be added to the carboxy terminus of the peptide.

Furthermore, the peptides of the invention may be synthesized to modify their configuration. For example, the D isomer of one or more amino acid residues of the peptide may be used instead of the usual L isomer. Furthermore, at least one of the amino acid residues of the peptides of the invention may be replaced with one of the well-known non-naturally occurring amino acid residues. Such modifications may help increase the stability, bioavailability and / or binding action of the peptides of the invention.

Similarly, the peptides or variants of the invention may be chemically modified by reacting specific amino acids either before or after peptide synthesis. Examples of such modifications are well known in the art and are incorporated herein by reference, for example, R.M. Lundblad, Chemical Requirements for Protein Modification, 3rd ed. It is summarized in CRC Press, 004 (Lundblad, 2004). Chemical modifications of amino acids include, but are not limited to, acylation, amidization, pyridoxylation of lysine, reduction alkylation, 2,4,6-trinitrobenzenesulfonic acid (TNBS). ) Trinitrobenzylation of amino groups, amide modification and sulfhydryl modification of carboxyl groups by perfirate oxidation of cysteine to cysteine acid, formation of mercury derivatives, mixed disulfide formation with other thiol compounds, reaction with maleimide, iodoacetic acid Alternatively, modification by carboxymethylation with iodoacetamide and carbamoylation with cyanate at alkaline pH is not limited to, but is not limited to. In this regard, one of ordinary skill in the art will discuss more detailed procedures for chemical modification of proteins in Chapter 15 of Current Protocols In Protein Science, Eds. Coligan et al. (John Wiley and Sons NY 1995-2000) (Coligan et al., 1995).

Briefly, for example, modification of arginyl residues in a protein is based on the reaction of adjacent dicarbonyl compounds such as phenylglyoxal, 2,3-butandione, and 1,2-cyclohexanedione to form an adduct. Often. Another example is the reaction of methylglyoxal with arginine residues. Cysteine can be modified without simultaneous modification of other nucleophilic sites such as lysine and histidine. As a result, numerous reagents are available for cysteine modification. Websites of companies such as Sigma-Aldrich (http://www.sigma-aldrich.com) provide information on specific reagents.

Selective reduction of disulfide bonds in proteins is also common. Disulfide bonds can be formed and oxidized during the heat treatment of biopharmacy. Certain glutamate residues may be modified using Woodward Reagent K. N- (3- (dimethylamino) propyl) -N'-ethylcarbodiimide can be utilized to form intramolecular crosslinks between lysine residues and glutamate residues. For example, diethylpyrocarbonate is a reagent for modifying histidyl residues in proteins. Histidine can also be modified using 4-hydroxy-2-nonenal. Lysine residues and other α-amino group reactants are useful, for example, in the binding of peptides to the surface or protein / peptide cross-linking. Lysine is the site of attachment of poly (ethylene) glycol and is the major modification site for protein glycosylation. Methionine residues in proteins can be modified, for example, with iodoacetamide, bromoethylamine, and chloramine-T.

Tyrosine residues can be modified using tetranitromethane and N-acetylimidazole. Crosslinking through the formation of dityrosine can be achieved by hydrogen peroxide / copper ions.

Recent studies on the modification of tryptophan include N-bromosuccinimid, 2-hydroxy-5-nitrobenzyl bromide or 3-bromo-3-methyl-2- (2-nitrophenyl mercapto) -3H-indole (BPNS). -Skatole) is used.

Successful modification of therapeutic proteins and peptides with PEG is often associated with prolonged circulating half-life, while cross-linking of proteins with glutaraldehyde, polyethylene glycol diacrylate, and formaldehyde is a hydrogel preparation. Used for. Chemical modification of allergens for immunotherapy is often achieved by carbamylation with potassium cyanate.

Peptides or variants comprising modified or non-peptide bonds are preferred embodiments of the invention. In general, peptides and variants (at least those containing peptide bonds between amino acid residues) are described in Lukas et al. It may be synthesized by Fmoc-polyamide mode solid phase peptide synthesis disclosed by (Lukas et al., 1981) and by the references cited therein. Temporary N-amino group protection is provided by the 9-fluorenylmethyloxycarbonyl (Fmoc) group. Repeated cleavage of this highly base-unstable protecting group is performed using 20% piperidine in N, N-dimethylformamide. Side chain functional groups are their butyl ether (for serine, threonine, and tyrosine), butyl ester (for glutamic acid and aspartic acid), butyloxycarbonyl derivative (for lysine and histidine), trityl derivative (for cysteine). , And 4-methoxy-2,3,6-trimethylbenzenesulfonyl derivative (in the case of arginine) may be protected. When glutamine or asparagine is a C-terminal residue, a 4,4'-dimethoxybenzhydryl group is utilized to protect the side chain amide functional group. The solid phase carrier is a polydimethyl-acrylamide polymer composed of three monomers, dimethylacrylamide (main chain monomer), bisacrylloylethylenediamine (crosslinking agent), and acryloylsarcosine methyl ester (functionalizing factor). Use as a base. The cleavable binding factor for the peptide-pair-resin used is an acid-labile 4-hydroxymethyl-phenoxyacetic acid derivative. With the exception of asparagine and glutamine added using the inverted N, N-dicyclohexyl-carbodiimide / 1 hydroxybenzotriazole-mediated conjugation procedure, all amino acid derivatives are added as their preformed symmetric anhydride derivatives. Will be done. All conjugation and deprotection reactions are monitored using ninhydrin, trinitrobenzene sulfonic acid or isatin test procedures. Upon completion of synthesis, the peptide is cleaved from the resin carrier and at the same time treatment with 95% trifluoroacetic acid containing a 50% scavenger mixture removes side chain protecting groups. Commonly used scavengers include ethanedithiol, phenol, anisole, and water, and the exact choice depends on the constituent amino acids of the peptide being synthesized. A combination of solid phase and solution phase methods for the synthesis of peptides is also possible (see, eg, (Bruckdorfer et al., 2004), and references cited therein).

Trifluoroacetic acid is removed by vacuum evaporation and subsequent grinding with diethyl ether results in a crude peptide. Any scavenger present is removed by a simple extraction procedure, which gives the scavenger-free crude peptide upon lyophilization of the aqueous phase. Reagents for peptide synthesis are usually available, for example, from Calbiochem-Novabiochem (Nottingham, UK).

Purification is optional in techniques such as recrystallization, size exclusion chromatography, ion exchange chromatography, hydrophobic interaction chromatography, and (usually) reverse phase high performance liquid chromatography using acetonitrile / water gradient separation. It may be carried out by one or a combination of.

Peptide analysis uses thin layer chromatography, electrophoresis, especially capillary electrophoresis, solid phase extraction (CSPE), reverse phase high performance liquid chromatography, and amino acid analysis after acid hydrolysis. It may be performed by FAB) mass spectroscopic analysis and by MALDI and ESI-Q-TOF mass spectroscopic analysis.

To select over-presented peptides, presentation profiles are calculated showing median sample presentations as well as repeat test variability. The profile juxtaposes a sample of the target tumor entity to the baseline of a normal sample. Then, by calculating the p-values of the linear mixed-effects model (Pinheroo et al., 2015) and correcting for multiple tests by false discovery rate (Benjamini and Hochberg, 1995), each of these profiles becomes an overpresented score. Can be integrated.

For HLA ligand identification and relative quantification by mass spectrometry, HLA molecules from shock-frozen samples were purified and HLA-related peptides were isolated. The isolated peptides were isolated and sequenced by online nanoelectrospray ionization (nanoESI) liquid chromatography-mass spectrometry (LC-MS) experiments. The resulting peptide sequence was confirmed by comparison of the fragmentation pattern of native TUMAP recorded from the CRC sample (N = 24A ^* 02 positive sample) with the fragmentation pattern of the corresponding synthetic standard peptide of the same sequence. rice field. Since the peptides were directly identified as ligands for HLA molecules in primary tumors, these results are the natural processing and presentation of the identified peptides on primary cancer tissues obtained from 24 CRC patients. Provide direct evidence of.

The discovery pipeline XPRESIDENT® v2.1 (see, eg, US Pat. No. 2013-096016, the entire contents of which are incorporated herein by reference) has several different non-existences. Allows identification and selection of reasonable over-presented peptide vaccine candidates based on direct relative quantification of HLA-binding peptide levels on cancer tissue compared to atrophic tissue and organs. It uses acquired LC-MS data processed in a proprietary data analysis pipeline for sequence identification algorithms, spectral clustering, ion counting, residence time alignment, charge state deconvolution, and normalization. Achieved by the development of unlabeled differential quantification, which combines.

Presentation levels were established, including error estimates for each peptide and sample. Peptides that are exclusively presented on tumor tissue, and peptides that are over-presented in tumor, have been identified in comparison to non-cancerous tissues and organs.

The HLA peptide complex from the CRC tissue sample was purified and the HLA binding peptide was isolated and analyzed by LC-MS (see Examples). All TUMAPs included in this application were identified on the primary CRC sample by this approach and their presentation on the primary CRC was confirmed.

TUMAPs identified on multiple CRCs and normal tissues were quantified using ion counting of unlabeled LC-MS data. The method assumes that the LC-MS signal area of the peptide correlates with its abundance in the sample. All quantitative signals of peptides in various LC-MS experiments were normalized based on central tendency, averaged per sample, and merged into a bar graph called a presentation profile. The presentation profile integrates different analytical methods such as protein database retrieval, spectral clustering, charge state deconvolution, and residence time alignment and normalization.

In addition, the discovery pipeline XPRESIDENT® v2. x allows direct absolute quantification of MHC-restricted, preferably HLA-restricted peptide levels on cancer or other infected tissue. Briefly, the total cell count was calculated from the total DNA content of the analyzed tissue sample. The total peptide amount of TUMAP in the tissue sample was measured by nano LC-MS / MS as a ratio of native TUMAP to a known amount of isotope-labeled version of TUMAP, the so-called internal standard. The efficiency of TUMAP isolation is that all selected TUMAP peptide: MHC complexes are added to the tissue lysate as early as possible in the TUMAP isolation procedure, followed by nanoLC following the completion of the peptide isolation procedure. -Determined by their detection by MS / MS. Total cell count and total peptide volume were calculated from triple measurements per tissue sample. Peptide isolation efficiencies were calculated as an average from 10 addition experiments, each measured in triplicate (see Examples 6 and Table 12).

The present invention provides peptides useful in treating cancer / tumor that present the peptides of the invention in excess or exclusively, preferably CRC. Mass spectrometry has shown that these peptides are naturally presented by HLA molecules on primary human CRC samples.

Many of the genes / proteins of origin from which the peptides are derived (also referred to as "full-length proteins" or "basal proteins") have been shown to be highly overexpressed in cancer compared to normal tissues. A high degree of tumor association of the gene of origin has been demonstrated, and "normal tissue" is intended to mean either healthy cells from the large intestine (colon or rectum) or other normal tissue cells in the context of the present invention. (See Example 2). In addition, the peptide itself is strongly over-presented on tumor tissue but not on normal tissue, and "tumor tissue" is a sample derived from a patient suffering from CRC in the context of the present invention. It shall mean (see Example 1).

HLA-binding peptides can be recognized by the immune system, especially T lymphocytes. T cells can destroy cells that present a recognized HLA / peptide complex, such as CRC cells that present an inducible peptide.

The peptides of the invention have been shown to be capable of stimulating T cell responses and / or being over-presented and can therefore be used for the production of TCRs such as antibodies and / or soluble TCRs according to the invention (implemented). See Example 3 and Example 4). In addition, peptides can also be used for the production of antibodies and / or TCRs, especially TCRs, according to the invention when complexed with their respective MHCs. Each method is well known to those of skill in the art and can also be found in the respective references. Thus, the peptides of the invention are useful in producing an immune response in a patient that can destroy tumor cells. The immune response in a patient encodes the peptide described, or a suitable precursor (eg, an elongated peptide, protein, or these peptides), ideally in combination with an immunogenicity-enhancing agent (ie, an adjuvant). It can be induced by administering the nucleic acid) directly to the patient. Since the target peptides of the invention are not presented in comparable numbers of copies on normal tissues, the immune response resulting from such therapeutic vaccination can be predicted to be highly specific for tumor cells. Prevents the risk of unwanted autoimmune reactions to the patient's normal cells.

The present specification further relates to the T cell receptor (TCR), which comprises a chain and an aβ chain (“α / βTCR”). Peptides capable of binding to TCRs and antibodies when presented by MHC molecules are also provided. The present specification also relates to nucleic acids, vectors, and host cells for expressing the TCRs and peptides herein; and methods of using them.

The term "T cell receptor" (abbreviated as TCR) refers to a heterodimer molecule comprising an α-polypeptide chain (α-chain) and a β-polypeptide chain (β-chain). The receptor can bind to the peptide antigen presented by the HLA molecule. The term also includes the so-called γ / δTCR.

In one embodiment, the present specification provides a method of producing a TCR as described herein, wherein the method is capable of expressing the TCR under conditions suitable for promoting the expression of the TCR, the host. It comprises the step of culturing the cells.

In another aspect herein, the antigen is on a class I or II MHC molecule that is expressed on the surface of the appropriate antigen presenting cell or artificial antigen presenting cell by contacting the antigen presenting cell with a sufficient amount of antigen. And by tetramerizing the antigen / class I or II MHC complex monomer, the antigen is loaded onto the class I or II MHC tetramer, according to the method described herein.

The α and β chains of α / βTCR and the γ and δ chains of γ / δTCR are generally considered to have two “regions”, namely a variable region and a stationary region, respectively. The variable region includes a connection of the variable region (V) and a connection region (J). The variable region may also include a leader region (L). The β and δ chains may also include the diversity region (D). The α and β constant regions may also include a C-terminal transmembrane (TM) region that anchors the α and β chains to the cell membrane.

With respect to γ / δTCR, the term “TCRγ variable region” refers to the connection between the TCRγV (TRGV) region and the TCRγJ (TRGJ) region without a leader region (L) in the usage herein, and the term TCRγ steady region is used. , Refers to the extracellular TRGC region, or refers to the C-terminal truncated TRGC sequence. Similarly, the term "TCRδ variable region" refers to the connection between the TCRδV (TRDV) region and the TCRδD / J (TRDD / TRDJ) region without a leader region (L), and the term "TCRδ constant region" is extracellular. Refers to the TRDC region, or refers to the C-terminal truncated TRDC sequence.

The TCRs herein preferably bind to the peptide-HLA molecular complex with a binding affinity (KD) of about 100 μM or less, about 50 μM or less, about 25 μM or less, or about 10 μM or less. More preferred is a high affinity TCR having a binding affinity of about 1 μM or less, about 100 nM or less, about 50 nM or less, and about 25 nM or less. Non-limiting examples of preferred binding affinity ranges for the TCRs of the invention are about 1 nM to about 10 nM; about 10 nM to about 20 nM; about 20 nM to about 30 nM; about 30 nM to about 40 nM; about 40 nM to about 50 nM; about 50 nM. Approximately 60 nM; about 60 nM to about 70 nM; about 70 nM to about 80 nM; about 80 nM to about 90 nM; and about 90 nM to about 100 nM.

In the usage herein, in the context of the TCRs herein, "specific binding" and their grammatical variants have a binding affinity (KD) of 100 μM or less for the peptide-HLA molecular complex. Used to mean having TCR.

The α / β heterodimer TCRs herein may have disulfide bonds introduced between their constant regions. Preferred TCRs of this type include those having a TRAC constant region sequence and a TRBC1 or TRBC2 constant region sequence, provided that Thr48 of TRAC and Ser57 of TRBC1 or TRBC2 are substituted with cysteine residues. The cysteine forms a disulfide bond between the TRAC constant region sequence of the TCR and the TRBC1 or TRBC2 constant region sequence.

In the presence or absence of the introduced interchain bonds described above, the α / β heterodimer TCR herein may have a TRAC constant region sequence and a TRBC1 or TRBC2 constant region sequence, the TCR. The TRAC constant region sequence and the TRBC1 or TRBC2 constant region sequence may be linked by a natural disulfide bond between Cys4 of exon 2 of TRAC and Cys2 of exon 2 of TRBC1 or TRBC2.

The TCR herein may include a detectable label selected from the group consisting of radionuclides, fluorophores, and biotin. The TCR herein may be conjugated to a therapeutically active agent such as a radionuclide, chemotherapeutic agent, or toxin.

In one embodiment, the TCR herein having at least one mutation in the α chain and / or at least one mutation in the β chain has modified glycosylation as compared to a non-mutated TCR.

In one embodiment, a TCR comprising at least one mutation in the TCRα chain and / or TCRβ chain comprises a non-variant TCRα chain and / or a non-variant TCRβ chain for a peptide-HLA molecular complex. Has at least 2-fold binding affinity and / or binding half-life. Increased affinity for tumor-specific TCRs and their utilization depend on the presence of a window of optimal TCR affinity. The presence of such a window is generally about one-tenth that of a TCR specific for an HLA-A2-restrictive pathogen compared to a TCR specific for an HLA-A2-restrictive tumor-associated autoantigen. Based on the observation that it has a KD value of. Tumor antigens may be immunogenic, but since tumors originate from the individual's own cells, it is now known that only mutant proteins or proteins with modified translational processing are considered foreign by the immune system. Has been done. Upregulated or overexpressed antigens (so-called self-antigens) do not necessarily induce a functional immune response to the tumor. T cells that express TCRs that are highly responsive to these antigens undergo a process known as central immune tolerance, in which only T cells with low affinity TCRs for self-antigens remain in the thymus. Is negatively selected in. Therefore, the affinity of the TCR or variant herein for the peptide according to the invention can be increased by methods well known in the art.

The present specification further relates to a method of identifying and isolating a TCR according to the present specification, wherein the method comprises ^{incubating PBMCs from healthy HLA-A *} 02 negative donors with A2 / peptide monomers and PBMCs. It comprises incubating with tetrameric phycoerythrin (PE) and isolating highly bound active T cells by fluorescence activated cell selection (FACS) Calibur analysis.

The present specification further relates to a method of identifying and isolating a TCR according to the present specification, wherein the T cell expresses a diverse human TCR repertoire that compensates for a mouse TCR deficiency, a whole human TCRαβ gene gene. A step of obtaining recombinant mice having loci (1.1 and 0.7 Mb), a step of immunizing the mice with the target peptide, and PBMCs obtained from the recombinant mice together with tetrameric phycoerythrin (PE). It comprises incubating and isolating highly bound active T cells by fluorescence activated cell selection (FACS) Calibur analysis.

In one aspect, in order to obtain T cells expressing the TCR herein, the nucleic acid encoding the TCR-α and / or TCR-β chain herein is expressed in an expression vector such as a gamma retrovirus or lentivirus. Be cloned. Recombinant viruses are generated and then tested for functions such as antigen specificity and functional binding activity. The final product aliquot is then used to transduce a target T cell population (generally purified from the patient's PBMC), which is propagated prior to infusion into the patient. In another aspect, TCR RNA is synthesized by techniques known in the art, such as, for example, an in vitro transcription system (sys-tems), in order to obtain T cells expressing the TCR herein. The TCR RNA synthesized in vitro is then introduced by electroporation into primary CD8 + T cells obtained from a healthy donor to reexpress tumor-specific TCR-α and / or TCR-β chains.

To increase expression, the nucleic acids encoding TCR herein are: retrovirus long-term repeat (LTR), cytomegalovirus (CMV), mouse stem cell virus (MSCV) U3, phosphoglycerate kinase (PGK), It may be operably linked to strong promoters such as β-actin, ubiquitin, and the Simian virus 40 (SV40) / CD43 composite promoter, elongation factor (EF) -1a, and spleen focus forming virus (SFFV) promoter. In a preferred embodiment, the promoter is heterologous to the nucleic acid expressed. In addition to a strong promoter, the TCR expression cassettes herein increase central polyprint tract (cPPT) (Follenzi et al., 2000), which promotes nuclear translocation of lentivirus constructs, and RNA stability. It may contain additional elements that may increase the expression of the introduced gene, such as Woodchuck hepatitis virus post-transcriptional regulator (wPRE) (Zuffery et al., 1999), thereby increasing the level of introduced gene expression. ..

The α and β chains of the TCR of the present invention may be encoded by nucleic acids in separate vectors or by polynucleotides in the same vector.

To achieve high levels of TCR surface expression, both the TCR-α and TCR-β chains of the introduced TCR need to be transcribed at high levels. To do so, the TCR-α and TCR-β chains herein may be cloned into bicistronic constructs within a single vector that have been shown to be able to overcome this obstacle. Since the TCR-α and TCR-β chains are produced from a single transcript that splits into two proteins during translation to ensure the formation of equimolar ratios of TCR-α and TCR-β chains, TCR-α And the use of ribosome entry sites within the viral sequence between the TCR-β chains results in coordinated expression of both chains (Schmitt et al. 2009).

The nucleic acid encoding the TCR herein may be codon-optimized for increased expression from the host cell. Duplication of the genetic code allows some amino acids to be encoded by more than one codon, but certain codons are more "optimal" than others due to the relative availability of compatible tRNAs as well as other factors. (Gustafsson et al., 2004). Modifying the TCR-α and TCR-β gene sequences so that each amino acid is encoded by the optimal codon for mammalian gene expression, as well as removing mRNA instability motifs or potential splice sites, is a TCR. It has been shown to significantly enhance -α and TCR-β gene expression (Scholten et al., 2006).

In addition, mismatching between the introduced TCR chain and the endogenous TCR chain can lead to the acquisition of specificity that poses a significant risk to autoimmunity. For example, the formation of a mixed TCR dimer may reduce the number of CD3 molecules available to form a properly paired TCR complex, thus increasing the functional binding activity of cells expressing the introduced TCR. It can be significantly reduced (Kuball et al., 2007).

To reduce mispair, the C-terminal region of the introduced TCR chain herein is modified to increase interchain affinity while reducing the ability of the introduced chain to pair with endogenous TCR. You may. These strategies replace the C-terminal regions of human TCR-α and TCR-β with their mouse counterparts (mousized C-terminal regions); second to both the TCR-α and TCR-β chains of the introduced TCR. By introducing a cysteine residue from the C-terminal region, a second interchain disulfide bond is formed (cysteine modification); the interaction residues within the TCR-α and TCR-β chain C-terminal regions are exchanged (Cysteine modification). "Knob in hole"); and may include directly fusing the variable regions of the TCR-α and TCR-β chains to CD3ζ (CD3ζ fusion) (Schmitt et al. 2009).

In one embodiment, the host cell is genetically engineered to express the TCR of this specification. In a preferred embodiment, the host cell is a human T cell or T cell progenitor cell. In some embodiments, T cells or T cell progenitor cells are obtained from a cancer patient. In other embodiments, T cells or T cell progenitor cells are obtained from healthy donors. The host cells herein can be allogeneic or autologous with respect to the patient being treated. In one embodiment, the host is a γ / δT cell transformed to express α / βTCR.

A "pharmaceutical composition" is a composition suitable for administration to humans in a medical setting. Preferably, the pharmaceutical composition is sterile and manufactured in accordance with GMP guidelines.

The pharmaceutical composition comprises a peptide in either free form or in the form of a pharmaceutically acceptable salt (see also above). As used herein, "pharmaceutically acceptable salt" refers to a derivative of the disclosed peptide, which is modified by producing an acidic or basic salt of the agent. For example, the acid salt is prepared from a free base, in which the neutral form of the drug typically has _{two neutral-NHs, with reaction to the appropriate acid.} Suitable acids for preparing acidic salts include, for example, acetic acid, propionic acid, glycolic acid, pyruvate, oxalic acid, malic acid, malonic acid, succinic acid, maleic acid, fumaric acid, tartrate acid, citric acid, benzoic acid. Both organic acids such as acids, silicic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid and salicylic acid, as well as inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid and phosphoric acid nitrate. Can be mentioned. Conversely, preparations for the basic salt of the acid moiety that may be present on the peptide use pharmaceutically acceptable bases such as sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide, trimethylamine and the like. Is prepared.

In a particularly preferred embodiment, the pharmaceutical composition comprises a peptide as a salt of acetic acid (acetate), trifluoroacetic acid or hydrochloric acid (chloride).

Preferably, the agent of the invention is an immunotherapeutic agent such as a vaccine. It can be applied directly to the patient, to the affected organ, or systemically, i. d. , I. m. , S. c. , I. p. , And i. v. A subpopulation of patient-derived immune cells that are administered or applied in vitro to cells derived from a patient or human cell line, which are subsequently administered to the patient or used in vitro, are selected. It may then be re-administered to the patient. When the nucleic acid is administered to the cell in vitro, it may be useful to transfect the cell so that it co-expresses an immunostimulatory cytokine such as interleukin 2. The peptide may be substantially pure, or used in combination with an immunostimulatory adjuvant (see below), or in combination with immunostimulatory cytokines, or may be administered by a suitable delivery system, such as, for example, liposomes. The peptide may also be conjugated to a suitable carrier such as keyhole limpet hemosinen (KLH) or mannan (see WO 95/18145 and (Longenecker et al., 1993)). .. The peptide may also be labeled, a fusion protein, or a hybrid molecule. The peptides whose sequences are described in the present invention are expected to stimulate CD4 or CD8 T cells. However, stimulation of CD8 T cells is more efficient in the presence of the aid provided by CD4 T helper cells. Thus, in MHC class I epitopes that stimulate CD8 T cells, the fusion partner or section of the hybrid molecule provides an epitope that appropriately stimulates CD4-positive T cells. CD4 and CD8 stimulating epitopes are well known in the art and include those identified in the present invention.

In one aspect, the vaccine comprises at least one peptide having the amino acid sequence set forth in SEQ ID NOs: 1 to SEQ ID NO: 191 and at least one additional peptide, preferably 2-50, more preferably 2-25. More preferably 2 to 20, most preferably 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or 18 peptides. It consists of. The peptide may be derived from one or more specific TAAs or may bind to MHC class I molecules.

A further aspect of the invention provides a nucleic acid (eg, a polynucleotide) that encodes a peptide or peptide variant of the invention. The polynucleotide may be, for example, either a single-stranded and / or double-stranded DNA, cDNA, PNA, RNA or a combination thereof, as long as it encodes a peptide, or, for example, a phosphorothioate main strand. It may be an unmodified or stabilized form of a polynucleotide, such as a polynucleotide having, which may or may not contain an intron. Of course, only peptides containing naturally occurring amino acid residues linked by naturally occurring peptide bonds can be encoded by the polynucleotide. Yet a further aspect of the invention provides an expression vector capable of expressing the polypeptide according to the invention.

Various methods have been developed for linking polynucleotides, especially DNA, to vectors, for example through complementary attachment ends. For example, a complementary homopolymer sequence can be added to a DNA fragment inserted into the vector DNA. The vector and the DNA fragment are then linked by hydrogen bonds between the complementary homopolymer tails to form a recombinant DNA molecule.

Synthetic linkers containing one or more restriction enzyme recognition sites provide an alternative method of linking a DNA fragment to a vector. Synthetic linkers containing a variety of restriction endonuclease sites are available from International Biotechnologies Inc. It is commercially available from several sources, including New Haven, CN, and USA.

The preferred method of modifying the DNA encoding the polypeptide of the invention is described in Saiki RK, et al. A polymerase chain reaction as disclosed in (Saiki et al., 1988) is used. This method may be used to introduce the DNA into the appropriate vector, eg, by modifying the appropriate restriction enzyme recognition site, or it may be used in other useful fashions known in the art. It may be used to qualify. If a viral vector is used, a poxvirus or adenoviral vector is preferred.

DNA (or RNA in the case of a retroviral vector) may then be expressed in a suitable host to produce a polypeptide comprising the peptide or variant of the invention. In this way, expression vectors may be constructed using the DNA encoding the peptides or variants of the invention according to known techniques appropriately modified in light of the teachings contained herein. Then, using it, suitable host cells are transformed for the expression and production of the polypeptides of the invention. Such techniques include, for example, US Pat. No. 4,440,859, US Pat. No. 4,530,901, US Pat. No. 4,582,800, US Pat. No. 4, 677,063, US Pat. No. 4,678,751, US Pat. No. 4,704,362, US Pat. No. 4,710,463, US Pat. No. 4,757, 006, US Pat. No. 4,766,075, and US Pat. No. 4,810,648.

The DNA encoding the polypeptide constituting the compound of the invention (or RNA in the case of a retroviral vector) may be linked to a wide variety of other DNA sequences for introduction into a suitable host. Companion DNA depends on the nature of the host, the mode of introduction of the DNA into the host, and whether maintenance or integration of episomes is desired.

Generally, DNA is inserted into an expression vector, such as a plasmid, in the proper direction and correct reading frame for expression. If desired, the DNA may be ligated to a suitable transcriptional and translational regulatory regulatory nucleotide sequence recognized by the desired host, but such regulation is generally available in expression vectors. The vector is then introduced into the host through standard techniques. In general, not all hosts are transformed with the vector. Therefore, it is necessary to select transformed host cells. One-choice technique involves incorporating into an expression vector a DNA sequence having any required regulator that encodes a selectable trait within transformed cells such as antibiotic resistance.

Alternatively, the gene for such selectable traits may be on another vector used to co-transform the desired host cell.

Next, in view of the teachings disclosed herein, host cells transformed with the recombinant DNA of the invention are cultured for a sufficient period of time under suitable conditions known to those of skill in the art. The polypeptide can be expressed and then recovered.

Bacteria (eg E. coli and Bacillus subtilis), yeast (eg Saccharomyces cerevisiae), filamentous fungi (eg Aspergillus), plant cells, animal cells, and insect cells. A large number of expression systems are known, including, preferably the expression system can be mammalian cells such as CHO cells available from ATCC Cell Biologic Collection.

A typical mammalian cell vector plasmid for constitutive expression comprises a CMV or SV40 promoter with a suitable poly A tail and a resistance marker such as neomycin. One example is pSVL available from Pharmacia, Piscataway, NJ, USA. An example of an inducible mammalian expression vector, pMSG, is also available from Pharmacia. Useful yeast plasmid vectors are pRS403-406 and pRS413-416, usually available from Stratagene Cloning Systems, La Jolla, CA 92037, USA. The plasmids pRS403, pRS404, pRS405, and pRS406 are yeast integration plasmids (Yips) and incorporate the yeast selectable markers HIS3, TRP1, LEU2, and URA3. The plasmid pRS413-416 is a yeast centromere plasmid (Ycps). CMV promoter-based vectors (eg, manufactured by Sigma-Aldrich) are transient or stable expression, intracytoplasmic expression or secretion, and N-terminal or C-terminal in various combinations of FRAG, 3xFLAG, c-myc or MAT. Provide labeling. These fusion proteins enable the detection, purification, and analysis of recombinant proteins. The double-labeled fusion provides flexibility in detection.

The potent human cytomegalovirus (CMV) promoter regulatory region drives constitutive protein expression levels to as high as 1 mg / L in COS cells. In less potent cell lines, protein levels are typically about 0.1 mg / L. The presence of the SV40 origin of replication results in high levels of DNA replication in SV40 replication-acceptable COS cells. The CMV vector may contain, for example, an origin of replication of pMB1 (a derivative of pBR322) in bacterial cells, a b-lactamase gene for ampicillin resistance selection in bacteria, hGH poly A, and an f1 origin. Vectors containing preprotrypsin leader (PPT) sequences can induce FRAG fusion protein secretion into culture medium for purification using anti-FRAG antibodies, resins, and plates. Other vectors and expression systems for use in a variety of host cells are well known in the art.

In another embodiment, two or more peptides or peptide variants of the invention are encoded and thus sequentially expressed (similar to the "adlay structure" construct). In doing so, the peptide or peptide variant may be linked or fused together by a series of linker amino acids, such as LLLLLL, or may be linked without any additional peptide between them. These constructs can also be used for the treatment of cancer and may induce an immune response involving both MHC I and MHC II.

The invention also relates to host cells transformed with the polynucleotide vector construct of the invention. Host cells can be either prokaryotes or eukaryotes. Bacterial cells may be preferred prokaryotic host cells in some situations, typically, for example, Bethesda Research Laboratories Inc. , E. coli DH5 strains available from Bethesda, MD, USA, and E. coli strains such as RR1 (ATCC number 31343) available from US Microbial Strain Conservation Agency (ATCC) Rockville, MD, USA. Is. Preferred eukaryotic host cells include vertebrate cells such as those derived from yeast, insect, and mammalian cells, preferably mouse, rat, monkey or human fibroblasts and colon cell lines. Yeast host cells include YPH499, YPH500, and YPH501, commonly available from Stratagene Cloning Systems, La Jolla, CA 92037, USA. Preferred mammalian host cells include Chinese hamster ovary (CHO) cells available as CCL61 from ATCC, NIH Swiss mouse germ cells NIH / 3T3 available as CRL1658 from ATCC, and monkey kidney-derived COS-1 cells available as CRL1650 from ATCC. And 293 cells, which are human embryo-derived kidney cells. A preferred insect cell is an Sf9 cell that can be transfected with a baculovirus expression vector. An overview of the selection of suitable host cells for expression can be found, for example, in the Paulina Balbas and Argelia Lorence textbooks "Methods in Molecular Biology Recombinant Gene Expression, Review, OnlineS, OneS. 262-9 and other literature known to those of skill in the art.

Transformation of a suitable cell host with the DNA construct of the present invention is typically achieved by well-known methods that depend on the type of vector used. For transformation of prokaryotic host cells, see, eg, Cohen et al. (Cohen et al., 1972) and (Green and Sambrook, 2012). Yeast cell transformation was performed by Sherman et al. (Sherman et al., 1986). The method of Beggs (Beggs, 1978) is also useful. For vertebrate cells, reagents useful for transfecting such cells, such as calcium phosphate and DEAE-dextran or liposome formulations, are available from Stratagene Cloning Systems, or Life Technologies Inc. , Gaithersburg, MD 20877, USA. Electric perforation is also useful for transforming and / or transfecting cells and is well known in the art of transforming yeast cells, bacterial cells, insect cells, and vertebrate cells.

Successfully transformed cells, i.e. cells containing the DNA constructs of the invention, can be identified by well-known techniques such as PCR. Alternatively, an antibody can be used to detect the presence of protein in the supernatant.

It will be appreciated that certain host cells of the invention, such as bacteria, yeast, and insect cells, are useful in the preparation of the peptides of the invention. However, other host cells may be useful in a particular treatment. For example, antigen presenting cells such as dendritic cells may be usefully used to express the peptides of the invention such that they may be loaded into the appropriate MHC molecule. Accordingly, the invention provides a host cell comprising the nucleic acid or expression vector according to the invention.

In a preferred embodiment, the host cell is an antigen presenting cell, particularly a dendritic cell or an antigen presenting cell. APC loaded with a recombinant fusion protein containing prostate acid phosphatase (PAP) was used by the US Food and Drug Administration (FDA) on April 20, 2010 to treat asymptomatic or microsymptomatic metastatic HRPC. (Ciploisel T) (Rini et al., 2006; Small et al., 2006).

A further aspect of the invention provides a method of producing a peptide or variant thereof comprising culturing a host cell and isolating the peptide from the host cell or culture medium thereof.

In another embodiment, the peptides, nucleic acids or expression vectors of the invention are used in medicine. For example, peptides or variants thereof can be injected intravenously (iv), subcutaneously (sc), intradermal (id), intraperitoneally (ip), intramuscularly. (Im) May be formulated for injection. The preferred method of peptide injection is s. c. , I. d. , I. p. , I. m. , And i. v. Can be mentioned. Preferred methods for DNA injection include i. d. , I. m. , S. c. , I. p. , And i. v. Can be mentioned. For example, a dose of 50 μg to 1.5 mg, preferably 125 μg to 500 μg of peptide or DNA may be administered, depending on the respective peptide or DNA. Dose in this range have been successfully used in previous trials (Walter et al., 2012).

The polynucleotide used for active vaccination may be substantially pure or may be contained in a suitable vector or delivery system. The nucleic acid may be DNA, cDNA, PNA, RNA or a combination thereof. Methods of designing and introducing such nucleic acids are well known in the art. For an overview, see, for example, Teufel et al. (Teufel et al., 2005). Although polynucleotide vaccines are easy to prepare, the mechanism of action of these vectors in inducing an immune response is not completely understood. Suitable vectors and delivery systems include viral DNA and / or RNA such as adenovirus, vaccinia virus, retrovirus, herpesvirus, adeno-associated virus, or hybrid-based systems containing two or more viral components. Can be mentioned. Non-viral delivery systems include cationic lipids and cationic polymers, which are well known in the field of DNA delivery technology. Physical delivery, such as through a "gene gun", may also be used. The peptide or peptide group encoded by the nucleic acid may be, for example, a fusion protein with an epitope that stimulates T cells in their respective reverse CDRs, as described above.

The agents of the invention may also include one or more adjuvants. The adjuvant is a substance that non-specifically promotes or enhances an immune response against an antigen mediated by an immune response (eg, CD8-positive T cells and helper T (TH) cells, and is therefore useful in the agents of the invention. Suitable adjuvants include 1018 ISS, aluminum salt, AMPLIVAX®, AS15, BCG, CP-870,893, CpG7909, CyaA, dSLIM, flaggerin or flaggerin-derived TLR5 ligand, FLT3 ligand, GM-. CSF, IC30, IC31, Imikimod (ALDARA®), Reshikimod, ImuFact IMP321, Interleukins such as IL-2, L-13 and IL-21, Interferon-α or -β or PEGylated derivatives thereof, IS Patch, ISS, ISCOMARTIX, ISCOM, JuvImmune®, LipoVac, MALP2, MF59, Monophosphoryl Lipide A, Montanide IMS1312, Montanide ISA206, Montanide ISA50V, Montanide ISA-51, Water-in-oil and oil-in-water emulsion, O -432, OM-174, OM-197-MP-EC, ONTAK, OspA, PepTel® vector system, poly (lactide coglycolide) [PLG] -based and dextran microparticles, talactoferline SRL172, virosomes and other viruses. Similar particles, YF-17D, VEGF traps, R848, β-glucan, Pam3Cys, Aquila's QS21 stimulon derived from saponin, mycobacterial extracts and synthetic bacterial cell wall mimetics, and Ribi's Detox or Quil or Superfos. Other proprietary adjuncts include, but are not limited to, adjuvants such as Freund or GM-CSF; some specific for dendritic cells and their preparations. Immunological adjuvants (eg, MF59) have been previously described (Allison and Krummel, 1995). Antigens may also be used. Some cytokines are dendritic cell lymphatic tissues (eg, TNF-). Affecting migration to efficient antigen-presenting cells for T lymphocytes (eg, GM-CSF, IL-1, IL-4) Accelerating dendritic cell maturation (US Pat. No. 5,849,589, the entire contents of which are specifically incorporated herein by reference), and immunopotentiators (eg, IL-12). , IL-15, IL-23, IL-7, IFN-α, IFN-β) and is directly associated with acting (Gabrilovich et al. , 1996).

CpG immunostimulatory oligonucleotides have also been reported to enhance adjuvant efficacy in a vaccine environment. Without being bound by theory, CpG oligonucleotides act by activation of the endogenous (non-adaptive) immune system through Toll-like receptors (TLRs), primarily TLR9. CpG-induced TLR9 activation includes a wide variety of antigens, including polysaccharide or protein antigens, live or dead viruses, dendritic cell vaccines, autologous vaccines, and polysaccharide conjugates in both prophylactic and therapeutic vaccines. Enhances antigen-specific humoral and cellular responses to. More importantly, it enhances dendritic cell maturation and differentiation, promotes TH1 cell activation, even in the absence of CD4 T cell assistance, and potent cytotoxic T lymphocytes ( CTL) results in production. The TH1 bias induced by TLR9 stimulation is usually maintained even in the presence of vaccine adjuvants such as alum or incomplete Freund's adjuvant (IFA), which promotes TH2 bias. CpG oligonucleotides still exhibit higher adjuvant activity when formulated or co-administered with other adjuvants, or in formulations such as microparticles, nanoparticles, lipid emulsions, or similar formulations. If the antigen is relatively weak, it is especially needed to induce a strong response. They also accelerate the immune response, allowing in some experiments an approximately double-digit reduction in antigen dose with an antibody response comparable to the total vaccine dose without CpG (Krieg, 2006). US Pat. No. 6,406,705B1 describes the combination of CpG oligonucleotides, non-nucleic acid adjuvants, and antigens to induce an antigen-specific immune response. The CpG TLR9 antagonist is a dSLIM (double stem-loop immunomodulator) manufactured by Mologen (Berlin, Germany), which is a preferred component of the pharmaceutical composition of the present invention. Other TLR-binding molecules such as RNA-binding TLR7, TLR8 and / or TLR9 may also be used.

Other examples of useful adjuvants include chemically modified CpG (eg, CpR, Idera); dsRNA analogs such as poly (I: C) and their derivatives (eg AmpliGen®, Hiltonol®, Poly). (ICLC), Poly (IC-R), Poly (I: C12U), Non-CpG Bacterial DNA or RNA; and Cyclophosphamide, Snitinib, Bevacizumab®, Celeblex, NCX-4016, Sildenafil, Tadalafil, Immunoactive small molecules and antibodies such as valdenafil, sorafenib, temozolomide, temsirolimus, XL-999, CP-547632, pazopanib, VEGF Trap, ZD2171, AZD2171, anti-CTLA4; other antibodies that target important structures of the immune system. (Eg, anti-CD40, anti-TGFβ, anti-TNFα receptor); SC58175, but not limited to, which may act therapeutically and / or as an adjuvant. The amounts and concentrations of adjuvants and additives useful in the above can be readily determined by those skilled in the art without undue experimentation.

Preferred adjuvants are anti-CD40, imiquimod, resiquimod, GM-CSF, cyclophosphamide, sunitinib, bevacizumab, interferon α, CpG oligonucleotides and derivatives, poly (I: C) and derivatives, RNA, sildenafil, and PLG or virosomes. It is a fine particle formulation.

In a preferred embodiment of the pharmaceutical composition according to the invention, the adjuvant comprises a group consisting of colony stimulating factors such as granulocyte macrophage colony stimulating factor (GM-CSF, sargramostim), cyclophosphamide, imiquimod, resiquimod, and interferon α. Be selected.

In a preferred embodiment of the pharmaceutical composition according to the invention, the adjuvant is selected from the group consisting of colony stimulating factors such as granulocyte macrophage colony stimulating factor (GM-CSF, sargramostim), cyclophosphamide, imiquimod, and resiquimod. .. In a preferred embodiment of the pharmaceutical composition according to the invention, the adjuvant is cyclophosphamide, imiquimod or resiquimod. Still more preferred adjuvants are Montandide IMS 1312, Montandide ISA 20, Montandide ISA 50V, Montandide ISA-51, Poly ICLC (Hiltonol®), and anti-CD 40 mAB or combinations thereof.

This composition is used for parenteral administration, such as subcutaneous, intradermal, intramuscular, etc., or for oral administration. To this end, the peptide and optionally other molecules are dissolved or suspended in a pharmaceutically acceptable, preferably aqueous carrier. In addition, the composition may contain excipients such as buffers, binders, blasting agents, diluents, flavors, lubricants and the like. Peptides can also be administered with immunostimulatory substances such as cytokines. A detailed list of excipients that can be used in such compositions is described, for example, in A.I. It can be adopted from Kibe, Handbook of Pharmaceutical Excipients (Kibbe, 2000). The composition can be used for the prevention, prevention and / or treatment of adenomatous or cancerous diseases. Exemplary formulations are, for example, in European Patent No. 211253.

It is important to understand that the immune response evoked by the vaccine according to the invention attacks cancers at different cell division stages and different stages of development. In addition, different cancer-related signaling pathways are attacked. This is an advantage over vaccines that may address only one or a few targets and cause easy adaptation of the tumor to attack (tumor escape). Moreover, not all individual tumors express the same pattern of antigens. Therefore, the combination of several tumor-related peptides ensures that every tumor has at least a portion of the target. The composition is designed with the expectation that each tumor will express some of the antigens and covers several independent pathways required for tumor growth and maintenance. Therefore, vaccines can be easily used "ready" for a larger patient population. This means that the preliminary selection of patients treated with the vaccine can be limited to HLA typing and does not require any additional biomarker assessment of antigen expression, but some targets are induced immunity. It is still certain that the response will be attacked simultaneously, which is important for efficacy (Banchereau et al., 2001; Walter et al., 2012).

As used herein, the term "scaffold" refers to a molecule that specifically binds to a (eg, antigenic) determinant. In one embodiment, the scaffold also has an entity to which it adheres (eg, the (second) antigen binding moiety), eg, an antigenic determinant (eg, a complex of peptide and MHC described in this application). It can be directed to specific tumor cells or type target sites such as tumor stroma. In another embodiment, the caffold can activate signal transduction via its target antigen, for example, a T cell receptor complex antigen. Scaffolds include antibodies and their fragments, antibody antigen binding domains comprising antibody heavy chain variable regions and antibody light chain variable regions, at least one ankyrin repeat motif and a single domain antigen binding (SDAB) molecule. Included, but not limited to, binding proteins, antigeners, (soluble) TCRs, and (modified) cells such as, but not limited to, allogeneic or autologous T cells. A binding assay can be performed to assess whether the molecule is a scaffold that binds to the target.

A "specific" binding is one in which a scaffold equipped with an active molecule capable of killing a cell carrying a specific target kills another cell that does not carry a specific target and presents another peptide-MHC complex. It means that the scaffold binds to the peptide-MHC-complex of interest even better than other native peptide-MHC-complexes to the extent that it cannot. If the peptide of the cross-reactive peptide-MHC is not naturally present, i.e., not derived from the human HLA-peptideome, binding to other peptide-MHC complexes is irrelevant. Tests for assessing target cell death are well known in the art. They should be performed using target cells (primary cells or cell lines) with unmodified peptide-MHC presentation, or cells loaded with peptides to reach naturally occurring peptide-MHC levels. be.

Each scaffold can include a label, which allows the binding scaffold to be detected by determining the presence or absence of the signal provided by the label. For example, the scaffold can be labeled with fluorescent dye or any other applicable cell marker molecule. Such marker molecules are well known in the art. For example, fluorescent labels provided by fluorescent dyes may provide visualization of bound aptamers by fluorescence or laser scanning microscopy or flow cytometry.

Each scaffold can be conjugated to a second active molecule such as IL-21, anti-CD3, anti-CD28.

For more information on polypeptide scaffolds, see, for example, the background section of WO 2014/071978A1 and the references cited therein.

The present invention further relates to aptamers. Aptamers (see, eg, WO 2014/191359, and the references cited therein) are short single-stranded nucleic acid molecules that are folded into a given three-dimensional structure and are unique. Can recognize target structures. They seemed to be a good alternative for developing targeted therapies. Aptamers have been shown to selectively bind to a variety of complex targets with high affinity and specificity.

Aptamers that recognize molecules located on the cell surface have been identified in the last decade and provide a means of developing diagnostic and therapeutic approaches. Aptamers have been shown to be nearly non-toxic and immunogenic, making them promising candidates for biomedical applications. Indeed, aptamers, such as, for example, prostate-specific membrane antigen recognition aptamers, have been successfully used for targeted therapies and have been shown to be able to function in xenograft in vivo models. In addition, aptamers that recognize specific tumor cell lines have been identified.

DNA aptamers can be selected to exhibit broad-spectrum recognition properties for a variety of cancer cells, especially those derived from solid tumors, while not recognizing nontumorogenic and primary healthy cells. If the identified aptamer not only recognizes a specific tumor subtype, but rather interacts with a series of tumors, this makes the aptamer applicable as a so-called broad spectrum diagnostic and therapeutic agent.

In addition, studies of cell binding behavior by flow cytometry showed that aptamers showed very good apparent affinity within the nanomolar concentration range.

Aptamers are useful for diagnostic and therapeutic purposes. Furthermore, it could be shown that some of the aptamers are taken up by tumor cells and thus can function as molecular vehicles for targeted delivery of anti-cancer agents such as siRNA into tumor cells.

Aptamers use cell SELEX (Systematic Evolution) technology against complex targets such as cells and tissues, and the sequences and MHC molecules set forth in any of SEQ ID NOs: 1 to 191 according to the invention. And can be selected, preferably for peptide complexes and the like.

The peptides of the invention can be used to generate and develop specific antibodies to the MHC / peptide complex. These can be used for treatments that target toxins or radioactive substances to the affected tissue. Another use of these antibodies could be the targeting of radionuclides for imaging purposes such as PET to affected tissue. This application can help detect small metastases, or determine the size and accurate location of diseased tissue.

Therefore, it is a further aspect of the invention to provide a method for producing a recombinant antibody that specifically binds to a human major tissue compatible complex (MHC) class I or II complexed with an HLA-binding antigen. The method is a soluble form of a genetically engineered non-human mammal complexed with the HLA-binding antigen comprising cells expressing the human major tissue compatible complex (MHC) class I or II. A phage display library that presents the protein molecule encoded by the mRNA molecule; with the step of immunizing with the MHC class I or II molecule of. The step of making; the step of isolating at least one phage from the phage display library comprises the step of isolating the at least one phage from the human major tissue compatible complex complexed with the HLA-binding antigen. The antibody is presented that specifically binds to body (MHC) class I or II.

It is also a further aspect of the invention to provide an antibody that specifically binds to a human major histocompatibility complex (MHC) class I or II complexed with an HLA-binding antigen, wherein the antibody. Preferred are polyclonal antibodies, monoclonal antibodies, bispecific antibodies and / or chimeric antibodies.

The respective methods for producing such antibodies and single-stranded Class I major histocompatibility complex, as well as other tools for producing these antibodies, are all by reference in their entirety for the purposes of the present invention. Explicitly incorporated, International Publication No. 03/068201, International Publication No. 2004/084798, International Publication No. 01/72768, International Publication No. 03/070752, and literature (Cohen et al. , 2003a; Cohen et al., 2003b; Denkberg et al., 2003).

Preferably, the antibody binds to the complex with a binding affinity of less than 20 nanomolar, preferably less than 10 nanomolar, which is also considered "specific" in the context of the invention.

The present invention is a peptide comprising a sequence selected from the group consisting of SEQ ID NOs: 1 to SEQ ID NO: 191 or a variant thereof that is at least 88% homologous (preferably identical) to SEQ ID NOs: 1 to SEQ ID NO: 191. Or with respect to its variants that cross-react T cells with the peptide, the peptide is not the underlying full-length polypeptide.

The present invention is a peptide comprising a sequence selected from the group consisting of SEQ ID NOs: 1 to SEQ ID NO: 191 or a variant thereof that is at least 88% homologous (preferably the same) as SEQ ID NO: 1 to SEQ ID NO: 191. Further, the peptide or variant has a full length of 8-100, preferably 8-30, most preferably 8-14 amino acids.

The present invention further relates to peptides according to the invention, which have the ability to bind to human major histocompatibility complex (MHC) class I or II molecules.

The present invention further relates to peptides according to the invention, wherein the peptide comprises or consists essentially of the amino acid sequences set forth in SEQ ID NOs: 1 to SEQ ID NO: 191.

The invention further relates to peptides according to the invention, wherein the peptide is (chemically) modified and / or contains non-peptide bonds.

The invention further relates to peptides according to the invention, wherein the peptide is part of a fusion protein, in particular contains an N-terminal amino acid of an HLA-DR antigen-related invariant chain (Ii), or the peptide is, for example, dendritic. Fuse to (and into) antibodies such as cell-specific antibodies.

The invention further relates to nucleic acids encoding peptides according to the invention, but the peptides are not fully (full length) human proteins.

The present invention further relates to nucleic acids according to the invention, which are DNA, cDNA, PNA, RNA or combinations thereof.

The present invention further relates to an expression vector capable of expressing the nucleic acid according to the present invention.

The invention further relates to peptides according to the invention, nucleic acids according to the invention or expression vectors according to the invention, which are used in medicine, especially in the treatment of CRC.

The invention further relates to a host cell comprising the nucleic acid according to the invention or the expression vector according to the invention.

The present invention further relates to antigen presenting cells, preferably host cells according to the invention, which are dendritic cells.

The present invention further relates to a method for producing a peptide according to the invention, comprising the steps of culturing a host cell according to the invention and isolating the peptide from the host cell or a culture medium thereof.

The present invention relates to a method according to the invention, wherein the antigen is loaded onto a Class I or II MHC molecule expressed on the surface of a suitable antigen presenting cell by contacting the antigen presenting cell with a sufficient amount of antigen. Further about.

The present invention further relates to a method according to the invention, wherein the antigen presenting cell comprises an expression vector capable of expressing the peptide, which comprises SEQ ID NO: 1 to SEQ ID NO: 191 or the different amino acid sequence.

The present invention further relates to activated T cells produced by the method according to the present invention, wherein the T cells selectively recognize cells that abnormally express a polypeptide comprising the amino acid sequence according to the present invention.

The present invention comprises killing a target cell in a patient that abnormally expresses a polypeptide comprising any amino acid sequence according to the invention, comprising the step of administering to the patient an effective number of T cells according to the invention. Regarding further.

The present invention relates to any of the peptides described, nucleic acids according to the invention, expression vectors according to the invention, cells according to the invention, or activated cytotoxic T lymphocytes according to the invention, as agents or in the manufacture of agents. , Further regarding use. The present invention further relates to use according to the present invention, wherein the agent is effective against cancer.

The present invention further relates to use according to the invention, wherein the agent is a vaccine. The present invention further relates to use according to the present invention, wherein the agent is effective against cancer.

Further according to the present invention, the cancer cells are CRC cells, and lung cancer, brain cancer, gastric cancer, kidney cancer, liver cancer, pancreatic cancer, prostate cancer, leukemia, breast cancer. , Mercel cell cancer, melanoma, ovarian cancer, and other solid or hematologic tumor cells such as esophageal cancer.

The invention further relates to specific peptide-based labeled proteins and biomarkers according to the invention, referred to herein as "targets," which can be used in the diagnosis and / or prognosis of CRC. The invention also relates to the use of these novel targets for the treatment of cancer.

The term "antibody" or "antibody" is used broadly herein and includes both polyclonal and monoclonal antibodies. In addition to untreated or "complete" immunoglobulin molecules, the term "antibody" refers to the desired properties according to the invention (eg, specific binding of CRC marker (poly) peptides, expression of cancer marker genes at increased levels. Delivering toxins to CRC cells and / or inhibiting the activity of CRC marker polypeptides) fragments (eg, CDRs, Fv, Fab, and Fc fragments), or immunoglobulin molecules thereof. And the immunoglobulin molecule humanized version of the polymer is also included.

Wherever possible, the antibodies of the invention may be purchased from commercial sources. Further, the antibody of the present invention may be produced by using a well-known method. Those of skill in the art will appreciate that either full-length CRC marker polypeptides or fragments thereof may be used to produce the antibodies of the invention. The polypeptide used to produce the antibodies of the invention may be partially or completely purified from natural sources, or may be produced using recombinant DNA technology.

For example, a cDNA encoding a peptide according to the invention, such as the peptide set forth in SEQ ID NOs: 1 to SEQ ID NO: 191; or variants or fragments thereof are prokaryotic cells (eg, bacteria) or eukaryotic cells (eg, yeast, etc.). A monoclonal or polyclonal antibody that can be expressed in an insect or mammalian cell) and then the recombinant protein is purified to specifically bind to a CRC marker polypeptide used to generate an antibody according to the invention. Can be used to produce formulations.

Those skilled in the art will find that the generation of two or more different sets of monoclonal or polyclonal antibodies is the specificity and affinity required for their intended use (eg, ELISA, immunohistochemical testing, in vivo imaging, immunotoxin therapy). You will understand that it maximizes the likelihood of obtaining an antibody with. Antibodies were tested for their desired activity by known methods according to the intended use of the antibody against it (eg, ELISA, immunohistochemical testing, immunotherapy, etc .; further guidance on antibody production and testing. For, see, for example, Greenfield, 2014 (Greenfield, 2014). For example, the antibody may be tested in an ELISA assay, Western blot, immunohistochemical staining of formalin-fixed cancer or frozen tissue sections. After their initial in vitro characterization, antibodies intended for therapeutic or in vivo diagnostic use are tested by known clinical trials.

The term "monoclonal antibody" as used herein refers to an antibody obtained from a substantially homogeneous population of antibodies; that is, the individual antibodies that make up the population may be present in trace amounts. It is the same except for spontaneous mutations. As used herein, "monoclonal antibodies" are those in which heavy and / or some of the light chains are derived from a particular species or of a particular antibody class or as long as they exhibit the desired antagonistic activity. While identical or homologous to the corresponding sequence in an antibody belonging to a subclass, the rest of the chain is identical or homologous to the corresponding sequence in an antibody derived from another species or belonging to another antibody class or subclass. Specific inclusion of a "chimeric" antibody, as well as fragments of such antibody (US Pat. No. 4,816,567, the entire contents of which are incorporated herein by reference).

The monoclonal antibody of the present invention may be prepared using the hybridoma method. In hybridoma methods, mice or other suitable host animals are typically immunized with an immune agent to produce lymphocytes that produce or can produce antibodies that specifically bind to the immune agent. Alternatively, lymphocytes may be immunized in vitro.

Monoclonal antibodies may also be produced by recombinant DNA methods such as those described in US Pat. No. 4,816,567. The DNA encoding the monoclonal antibody of the invention can be readily obtained using conventional procedures (eg, by using an oligonucleotide probe that can specifically bind to the genes encoding the heavy and light chains of the mouse antibody). Can be isolated and sequenced.

The in vitro method is also suitable for preparing monovalent antibodies. Digestion of antibodies to make antibody fragments, in particular Fab fragments, can be accomplished using conventional techniques known in the art. For example, digestion can be performed using papain. Examples of papain digestion are described in International Publication No. 94/29348 and US Pat. No. 4,342,566. Papain digestion of an antibody typically results in two identical antigen-binding fragments, each referred to as a Fab fragment, with a single antigen-binding site and the remaining Fc fragment. Pepsin treatment results in F (ab') 2 and pFc' fragments.

An antibody fragment, whether attached to other sequences or not, is a specific region or amino acid residue provided that the activity of the fragment is not significantly altered or impaired as compared to an unmodified antibody or antibody fragment. Can also include insertions, deletions, substitutions, or other selected modifications of. These modifications may provide several additional properties such as removal / addition of amino acids capable of disulfide bonds, increased biolifespan thereof, and modification of their secretory properties. In any case, the antibody fragment must have bioactive properties such as binding activity, binding regulation in the binding region. Functional or active regions of an antibody may be identified by mutagenesis of specific regions of the protein followed by expression and testing of the expressed polypeptide. Such methods are readily apparent to skilled practitioners in the art and may include site-directed mutagenesis of the nucleic acid encoding the antibody fragment.

The antibody of the present invention may further comprise a humanized antibody or a human antibody. A humanized form, such as a non-human (eg, mouse) antibody, is a chimeric immunoglobulin, an immunoglobulin chain or fragment thereof (Fv, Fab, Fab'or other antigen of the antibody, containing the smallest sequence derived from a non-human immunoglobulin. Combined subarray, etc.). As humanized antibodies, non-human species such as mice, rats or rabbits in which the residues from the complementarity determining regions (CDRs) of the recipient have the desired specificity, affinity, and ability. Human immunoglobulins (recipient antibodies) that are replaced by residues from the CDRs of the donor antibody). In some cases, the Fv framework (FR) residues of human immunoglobulin are replaced by the corresponding non-human residues. Humanized antibodies may also contain residues that are not found in either the recipient antibody or the imported CDR or framework sequence. In general, humanized antibodies consist of substantially all of at least one and typically two variable regions, in which all or substantially all of the CDR regions are those of non-human immunoglobulins. Correspondingly, all or substantially all of the FR regions are of the human immunoglobulin common sequence. Humanized antibodies optimally also comprise at least a portion of an immunoglobulin constant region (Fc), typically a human immunoglobulin constant region.

Methods of humanizing non-human antibodies are well known in the art. Usually, humanized antibodies have one or more amino acid residues introduced from non-human origin. These non-human amino acid residues are often referred to as "introduced" residues, which are typically obtained from the "introduced" variable region. Humanization can be essentially performed by substituting the rodent CDR (s) or CDR (singular) sequences with the corresponding human antibody sequences. Thus, such "humanized" antibodies are chimeric antibodies (US Pat. No. 4,816,567), in which substantially less than the intact human variable region is from non-human species. Replaced by the corresponding sequence of. In practice, humanized antibodies are typically human antibodies, in which some CDR residues and possibly some FR residues are due to residues from similar sites in the rodent antibody. Will be replaced.

For immunization, transgenic animals (eg, mice) capable of producing a complete repertoire of human antibodies in the absence of endogenous immunoglobulin production can be used. For example, homozygous deletions of antibody heavy chain linking region genes in chimeric and germline mutant mice have been described to result in complete inhibition of endogenous antibody production. Transcribing a human germline immunoglobulin gene array in such germline mutant mice results in the production of human antibodies during antigen challenge. Human antibodies can also be produced in phage display libraries.

The antibodies of the invention are administered to the subject, preferably in a pharmaceutically acceptable carrier. Typically, an appropriate amount of a pharmacologically acceptable salt is used in the formulation to make the formulation isotonic. Examples of pharmacologically acceptable carriers include saline, Ringer's solution, and dextrose solution. The pH of the solution is preferably about 5 to about 8, more preferably about 7 to about 7.5. Further carriers include semipermeable matrix sustained release formulations of solid hydrophobic polymers containing antibodies, the matrix in the form of shaped articles such as, for example, films, liposomes or microparticles. It will be apparent to those of skill in the art that certain carriers may be more preferred, for example, depending on the route of administration and concentration of the antibody being administered.

Antibodies are administered to a subject, patient, or cell by injection (eg, intravenously, intraperitoneally, subcutaneously, intramuscularly) or by other means such as infusion to ensure delivery to the bloodstream in its effective form. Can be administered. Antibodies may also be administered by intratumoral or peritumor routes to exert topical and systemic therapeutic effects. Local or intravenous injections are preferred.

Effective doses and schedules for administering the antibody may be determined empirically, and the implementation of such measurements is within the skill of the art. Those skilled in the art will understand that the antibody dose that must be administered will vary, for example, depending on the subject to whom the antibody is administered, the route of administration, the particular antibody type used, and other agents administered. There will be. Typical daily doses of antibodies used alone may range from about 1 (μg / kg to up to 100 mg / kg body weight or more) per day, depending on the factors mentioned above, preferably treating CRC. Following antibody administration, the efficacy of the therapeutic antibody can be assessed in a variety of ways well known to skilled practitioners, for example, receiving treatment using standard tumor imaging techniques. The size, number, and / or distribution of cancer in a subject may be monitored. It arrests tumor growth, results in tumor contraction, and / or compared to the course of disease that would occur in the absence of antibody administration. Antibodies administered therapeutically that prevent the development of new tumors are effective antibodies for the treatment of cancer.

It is also a further aspect of the invention to provide a method of producing a soluble T cell receptor (sTCR) that recognizes a specific peptide-MHC complex. Such soluble T cell receptors can be generated from specific T cell clones and their affinity can be enhanced by mutagenesis targeting complementarity determining regions. Phage displays can be utilized for the purpose of selecting T cell receptors (US Pat. No. 2010/0113300, (Lidy et al., 2012)). For the purpose of stabilizing T cell receptors in phage display and when used as a drug, for example, unnatural disulfide bonds, other covalent bonds (single-stranded T cell receptors), or dimerization. Depending on the domain, α and β chains can be linked (Boulter et al., 2003; Card et al., 2004; Willcox et al., 1999). T-cell receptors are escalating effector cells such as toxins, drugs, cytokines (see, eg, US Pat. No. 2013/0115191), anti-CD3 domains to exert specific functions on target cells. It can be linked to a domain or the like. Furthermore, it can be expressed in T cells used for adoptive immunity transmission. Further information can be found in International Publication No. 2004/033685A1 Pamphlet and International Publication No. 2004/074322A1 Pamphlet. The combination of TCRs is described in International Publication No. 2012-05647A1 Pamphlet. Further manufacturing methods are disclosed in International Publication No. 2013/057586A1 pamphlet.

In addition, the peptides and / or TCRs or antibodies or other binding molecules of the invention can be used to confirm a pathologist's biopsy sample-based cancer diagnosis.

Antibodies or TCRs may also be used for in vivo diagnostic assays. ^{Antibodies are usually on radioactive nucleotides (such as 111} In, ⁹⁹ Tc, ¹⁴ C, ¹³¹ I, ³ H, ³² P or ³⁵ S) so that the tumor can be located using immunoscintigraphy. Will be labeled. In one embodiment, the antibody or fragment thereof binds to the extracellular domain of two or more targets of a protein selected from the group consisting of the proteins described above, and has an affinity (Kd) of less than 1 × 10 μM.

Diagnostic antibodies may be labeled with probes suitable for detection by various imaging methods. Methods of detecting the probe include, but are limited to, fluorescence, optics, cofocal and electron microscopy; magnetic resonance imaging and spectroscopy; fluoroscopy, computed tomography, and positron emission tomography. It's not a thing. Suitable probes include, but are limited to, fluorescein, rhodamine, eodin and other fluorophores, radioisotopes, gold, gadrinium and other lanthanides, paramagnetic iron, fluorine-18 and other positron emitting radionuclides. It is not something that will be done. In addition, the probe may be bifunctional or polyfunctional and may be detectable by two or more of the listed methods. These antibodies may be labeled directly or indirectly with the probe. Particularly well-approved attachment of a probe to an antibody includes covalent attachment of the probe, incorporation of the probe into the antibody, and covalent attachment of a chelating compound for probe binding. For immunohistochemical examination, diseased tissue samples may be fresh or frozen, or paraffin-embedded and fixed with a preservative such as formalin. Fixed or embedded sections containing the sample are contacted with labeled primary and secondary antibodies and in-situ protein expression is detected using the antibody.

Another aspect of the invention comprises an in vitro method for producing activated T cells, wherein the method comprises in vitro T cells to an antigen-loaded human MHC molecule expressed on the surface of a suitable antigen presenting cell. The antigen comprises the step of contacting for a sufficient time to activate in an antigen-specific manner, the antigen being the peptide according to the invention. Preferably, a sufficient amount of antigen is used with the antigen presenting cells.

Preferably, the mammalian cells have no or reduced levels or function of the TAP peptide transporter. Suitable cells lacking the TAP peptide transporter include T2, RMA-S, and Drosophila cells. TAP is a transporter associated with antigen processing.

Human peptide-loaded deficient cell line T2 is available under Catalog No. CRL1992 from the US Microbial Strain Conservation Agency of 12301 Parklawn Drive, Rockville, Maryland 20852, USA; Drosophila cell line Schneider Strain 2 is ATCC under Catalog No. CRL1963. Available from; mouse RMA-S cell lines are available from Ljunggren et al. (Ljunggren and Karre, 1985).

Preferably, prior to transfer, the host cell does not substantially express the MHC class I molecule. It is also preferred that the stimulator cells express important molecules in providing a co-stimulatory signal for T cells such as any of B7.1, B7.2, ICAM-1, and LFA3. Nucleic acid sequences of numerous MHC class I and co-stimulator molecules are publicly available from the GenBank and EMBL databases.

When the MHC class I epitope is used as an antigen, the T cells are CD8 positive T cells.

When antigen-presenting cells are transfected to express such epitopes, the cells preferably comprise an expression vector capable of expressing a peptide containing SEQ ID NOs: 1 to SEQ ID NO: 191 or a mutant amino acid sequence thereof. It consists of.

Several other methods may be used to produce T cells in vitro. For example, autologous tumor infiltrative lymphocytes can be used to generate CTLs. Plebanski et al. (Plebanski et al., 1995) utilized autologous peripheral blood lymphocytes (PLBs) in the preparation of T cells. In addition, autologous T cells can also be produced by pulsing dendritic cells with a peptide or polypeptide, or by infecting them with a recombinant virus. B cells can also be used in the production of autologous T cells. In addition, macrophages pulsed with peptides or polypeptides or infected with recombinant virus may be used in the preparation of self-CTL. S. Walter et al. (Walter et al., 2003) describe in vitro priming of T cells using artificial antigen presenting cells (aAPC), which is also a suitable method for producing T cells for selected peptides. .. In the present invention, aAPC was prepared by conjugating a preformed MHC: peptide complex to surface polystyrene particles (microbeads) by biotin: streptavidin biochemistry. This system allows accurate regulation of MHC densities on aAPC, which allows highly efficient, high or low binding activity antigen-specific T cell responses to be selectively elicited from blood samples. In addition to MHC: peptide complexes, aAPC should carry other proteins with costimulatory activity, such as anti-CD28 antibodies, that are conjugated to their surface. Moreover, such aAPC-based systems often require the addition of suitable soluble factors, such as cytokine-like interleukin 12.

Allogeneic cells may also be used in the preparation of T cells, the method being detailed in WO 97/26328, which is incorporated herein by reference. For example, in addition to Drosophila cells and T2 cells, other cells may be used to present antigens such as CHO cells, baculovirus-infected insect cells, bacteria, yeast, vaccinia infection target cells. In addition, plant viruses may be used (see, eg, Porta et al. (1994) describing the development of cowpea mosaic virus as a high yield system for the presentation of foreign peptides).

Activated T cells directed to the peptides of the invention are useful in therapeutic methods. Accordingly, a further aspect of the invention provides activated T cells available by the method of the invention described above.

Activated T cells produced by the above method selectively recognize cells that abnormally express a polypeptide comprising the amino acid sequences of SEQ ID NOs: 1 to SEQ ID NO: 191.

Preferably, the T cell recognizes the cell by interacting with the HLA / peptide complex (eg, binding) through its TCR. T cells are useful in a method of killing a target cell in a patient in which the target cell abnormally expresses a polypeptide comprising the amino acid sequence of the present invention, and the patient has an effective number of activated T cells. Be administered. The T cells administered to the patient may be derived from the patient and activated as described above (ie, they are autologous T cells). Alternatively, T cells come from another individual rather than the patient. Of course, if the individual is a healthy person, that is preferable. By "healthy individuals", the inventors are generally in good health, preferably have a competent immune system, and more preferably do not suffer from any disease that can be easily tested and detected. Means that.

In vivo, the target cells of CD8-positive T cells according to the invention can be tumor cells (sometimes expressing MHC class II) and / or (sometimes also expressing MHC class II; (Dengjell et al.). , 2006)) Can be stromal cells around the tumor (tumor cell).

The T cells of the present invention may be used as an active ingredient in a therapeutic composition. Accordingly, the invention also provides a method of killing a target cell in a patient in which the target cell abnormally expresses a polypeptide comprising the amino acid sequence of the invention, as defined above. It comprises the step of administering to the patient an effective number of T cells.

By "abnormally expressed", we are overexpressing the polypeptide compared to normal expression levels, or the gene is silent in the tissue from which the tumor is derived, but the tumor. It also means that it is expressed in. By "overexpression", we show that the polypeptide is at a level at least 1.2 times the level present in normal tissue; preferably at least twice the level present in normal tissue, more preferably at least 5. It means that it exists at double or ten times the level.

T cells may be obtained by methods known in the art, such as those described above.

Protocols for this so-called adoptive immunity transmission of T cells are well known in the art. For an overview, see Gattioni et al. and Morgan et al. (Gattinoni et al., 2006; Morgan et al., 2006).

Another aspect of the invention comprises the use of a peptide complexing with MHC to generate a T cell receptor on which the nucleic acid is cloned and introduced into a host cell, preferably a T cell. The genetically engineered T cells can then be transferred to the patient for cancer treatment.

Any molecule of the invention, namely peptides, nucleic acids, antibodies, expression vectors, cells, activated T cells, T cell receptors or nucleic acids encoding them, can be used to treat disorders characterized by cells that escape the immune response. It is useful. Thus, any molecule of the invention may be used as a drug or in the manufacture of a drug. Molecules may be used alone or in combination with other molecules of the invention or known molecules.

The present invention
(A) A container containing the above-mentioned pharmaceutical composition in solution or in lyophilized form;
(B) Optionally a second container containing a diluent or reconstitution solution for the lyophilized formulation; and (c) optionally, (i) use of the solution or (ii) lyophilization. Further objects are kits comprising instructions for reconstitution and / or use of the pharmaceutical product.

The kit may further include one or more of (iii) buffer, (iv) diluent, (V) filtration, (vi) needle, or (V) syringe. The container is preferably a bottle, vial, syringe or test tube; it may be a multi-use container. The pharmaceutical composition is preferably lyophilized.

The kit of the present invention preferably comprises the lyophilized formulation of the present invention in a suitable container and instructions for reconstitution and / or use thereof. Suitable containers include, for example, bottles, vials (eg, double chamber vials), syringes (such as double chamber syringes), and test tubes. The container may be made of a variety of materials such as glass or plastic. Preferably, the kit and / or container comprises instructions on or accompanying the container, which indicate reconfiguration and / or instructions for use. For example, the label may indicate that the lyophilized formulation is reconstituted to peptide concentrations as described above. The label may further indicate that the formulation is useful for subcutaneous administration or is for subcutaneous administration.

The container containing the product may be a multi-use vial, which allows repeated administration of the reconstituted product (eg, 2 to 6 doses). The kit may further include a second container comprising a suitable diluent (eg, sodium bicarbonate solution).

When the diluent and the lyophilized product are mixed, the final peptide concentration in the reconstituted product is preferably at least 0.15 mg / mL / peptide (= 75 μg), preferably 3 mg / mL / peptide (= 1500 μg) or less. be. The kit may further include other items desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, and package inserts with instructions.

The kit of the present invention comprises a single container containing the pharmaceutical composition formulation according to the invention to which other components (eg, other compounds or pharmaceutical compositions of these other compounds) have been added or not added. It may be used or it may have a separate container for each component.

Preferably, the kit of the present invention comprises a second compound (such as an adjuvant (eg, GM-CSF), chemotherapeutic agent, natural product, hormone or antagonist, antiangiogenic factor or inhibitor, apoptosis inducer or chelating agent). Or include the formulations of the invention packaged for use in conjunction with co-administration of the pharmaceutical composition. The components of the kit may be premixed, or each component may be in a separate and different container prior to administration to the patient. The components of the kit may be provided in one or more liquid solutions, preferably aqueous solutions, more preferably sterile aqueous solutions. The components of the kit may also be provided as a solid, which may be converted to a liquid by the addition of a suitable solvent, preferably provided in another different container.

The container of the treatment kit may be a vial, a test tube, a flask, a bottle, a syringe, or any other means of enclosing a solid or liquid. Usually, when there are two or more components, the kit contains a second vial or other container to allow separate dosing. The kit may also contain another container for a pharmaceutically acceptable liquid. Preferably, the therapeutic kit contains a device (eg, one or more needles, syringes, eye drops, pipettes, etc.) to allow administration of the agents of the invention that are components of the kit. do.

This product is suitable for peptide administration by any acceptable means such as oral (intestinal), nasal, ocular, subcutaneous, intradermal, intramuscular, intravenous or transdermal. Preferably, the administration is s. c. Most preferably i. d. It is an administration, and may be an administration by an infusion pump.

Since the peptides of the invention have been isolated from the CRC, the agents of the invention are preferably used to treat the CRC.

The present invention further relates to a method of producing a personalized drug for an individual patient, comprising the step of producing a pharmaceutical composition comprising at least one peptide selected from the reservoir of pre-screening TUMAP. , At least one peptide used in the pharmaceutical composition is selected for suitability in an individual patient. In one embodiment, the pharmaceutical composition is a vaccine. The method can also be adapted for downstream applications such as TCR isolation or soluble antibodies, and for producing T cell clones for other treatment options.

"Personalized medicines" are used solely for the treatment of such individual patients, including aggressive personalized cancer vaccines and adoptive cell therapy using autologous patient tissue, for a single patient. It shall mean a treatment method specifically adjusted for this purpose.

As used herein, the term "reservoir" refers to a group or set of peptides that have been pre-screened for immunogenicity and / or overpresentation in a particular tumor type. The term "reservoir" is not intended to imply that the particular peptide contained in the vaccine is pre-made and stored in physical equipment, but the possibility is also considered. It is explicitly considered that the peptide may be freshly prepared or pre-made and stored for each individualized vaccine produced. The reservoir (eg, the form of the database) is composed of tumor-related peptides that are highly overexpressed in the tumor tissue of CRC patients with various HLA-AHLA-B and HLA-C alleles. It may contain MHC class I and MHC class II peptides or extended MHC class I peptides. In addition to tumor-related peptides taken from several CRC tissues, the reservoir may contain HLA-A ^* 02 and HLA-A ^* 24-labeled peptides. These peptides allow quantitative comparison of the magnitude of T cell immunity induced by TUMAP and thus lead to important conclusions about the ability of the vaccine to elicit an antitumor response. Second, they serve as important positive control peptides derived from the "non-self" antigen in patients where no vaccine-induced T cell response to TUMAP derived from the "self" antigen is observed. Third, it may allow conclusions regarding the patient's immune capacity status.

TUMAPs for storage are identified using an integrated genomic function analysis approach (XPresident®) that combines gene expression analysis, mass spectrometry, and T cell immunology. The approach ensures that only TUMAP, which is truly present on a high proportion of tumors but is not or minimally expressed on normal tissue, is selected for further analysis. For initial peptide selection, CRC samples from patients and blood from healthy donors were analyzed in a stepwise approach:
1. 1. HLA ligands from malignant substances were identified by mass spectrometry. 2. Genome-scale messenger ribonucleic acid (mRNA) expression analysis was used to identify gene overexpression in malignant tissue (CRC) compared to a series of normal organs and tissues. The identified HLA ligands were compared to gene expression data. Preferably, the peptide over-presented or selectively presented on the tumor tissue, encoded by the selectively expressed or over-expressed gene as detected in step 2, is for the multiplex peptide vaccine. It was considered a suitable TUMAP candidate.

4. 5. A literature search was conducted to identify additional evidence supporting the validity of the identified peptide as a TUMAP. The association of overexpression at the mRNA level was confirmed by the re-detection of selected TUMAPs from step 3 on tumor tissue and the lack (or rare) detection of detection in healthy tissue.

6. In vitro immunogenicity assays were performed using human T cells from healthy donors as well as CRC patients to assess whether the selected peptides could induce in vivo T cell responses.

In one aspect, the peptide is pre-screened for immunogenicity prior to inclusion in storage. As an unintended example, the immunogenicity of the peptides contained in the reservoir was through repeated stimulation of CD8 + T cells from healthy donors by artificial antigen presenting cells loaded with peptide / MHC complex and anti-CD28 antibody. Determined by a method comprising in vitro T cell priming.

This method is preferred for rare cancers and for patients with rare expression profiles. In contrast to the multi-peptide mixture with a constant composition, the reservoirs currently being developed allow a significantly higher match between the actual expression of the antigen in the tumor and the vaccine. The multi-target approach utilizes several selected single or combined "off-the-shelf" peptides for each patient. In theory, an approach based on the selection of 5 different antigenic peptides, for example from a library of 50 antigenic peptides, alone yields approximately 17 million possible pharmaceutical (DP) compositions.

In one aspect, peptides are selected for inclusion in vaccines based on their suitability for individual patients as described herein or based on the methods according to the invention as described below.

HLA phenotype, transcriptmix, and peptide mix data are collected from the patient's tumor material and blood samples and are most appropriate for each patient and contain a "reservoir" and a patient-specific (ie, variant) TUMAP. Peptides are identified. Peptides that are selectively or overexpressed in a patient's tumor and, if possible, tested with the patient's individual PBMCs to exhibit strong in vitro immunogenicity are selected.

Preferably, the peptides included in the vaccine are (a) the step of identifying the tumor-related peptide (TUMAP) presented by the tumor sample from an individual patient; (b) the peptide identified in (a) above. It is identified by a method comprising comparing with a peptide reservoir; (c) selecting at least one peptide from the reservoir (database) associated with the tumor-related peptide identified in the patient. For example, the TUMAP presented by the tumor sample is (a1) overexpressed in the tumor sample by comparing the expression data from the tumor sample with the expression data from the normal tissue sample corresponding to the histological type of the tumor sample. Or with the step of identifying abnormally expressed proteins; (a2) Overexpression by the tumor by correlating the expression data with the sequence of MHC ligands bound to MHC class I and / or class II molecules in the tumor sample. It is identified by the step of identifying MHC ligands derived from proteins that are or are abnormally expressed. Preferably, the sequence of the MHC ligand is identified by eluting the binding peptide from the MHC molecule isolated from the tumor sample and sequencing the eluted ligand. Preferably, the tumor sample and normal tissue are obtained from the same patient.

In addition to or as an alternative to selecting peptides using a storage (database) model, TUMAP may be newly identified in a patient and then included in the vaccine. As an example, (a1) the expression data from the tumor sample is compared with the expression data from the normal tissue sample corresponding to the histological type of the tumor sample, and is overexpressed or abnormally expressed in the tumor sample. Steps to identify the protein; (a2) Correlate the expression data with the sequence of MHC ligands bound to MHC class I and / or class II molecules in the tumor sample and are overexpressed or abnormally expressed by the tumor. Candidate TUMAPs may be identified in a patient by the step of identifying an MHC ligand derived from a protein. As another example, a protein containing a mutation specific to a tumor sample may be identified as compared to a normal corresponding tissue from an individual patient, and a TUMAP specifically targeting the mutation may be identified. .. For example, the tumor genome, and the corresponding normal tissue genome, can be sequenced by whole genome sequencing. Genomic DNA and RNA are extracted from tumor tissue and normal non-mutated genomic germline DNA is extracted from peripheral blood mononuclear cells (PBMCs) to detect non-synonymous mutations in the protein coding region of the gene. The NGS approach applied is limited to the rearrangement of protein coding regions (exosome rearrangement). For this purpose, exon DNA from human samples is captured using a target enrichment kit provided by the supplier, followed by sequencing, for example by HiSeq2000 (Illumina). In addition, tumor mRNA is sequenced for direct quantification of gene expression and validation of the expression of the mutant gene in the patient's tumor. The resulting millions of sequence reads are processed through software algorithms. The output list includes mutations and gene expression. Tumor-specific somatic mutations are determined and prioritized by comparison with the diversity of PBMC-derived germline. The newly identified peptide can then be tested for immunogenicity as described above for the reservoir, and candidate TUMAPs that retain adequate immunogenicity are selected for inclusion in the vaccine.

In one exemplary embodiment, the peptides included in the vaccine are (a) a step of identifying a tumor-related peptide (TUMAP) presented by a tumor sample from an individual patient by the method described above; (b). () A step comparing the peptide identified in a) with a reservoir of preselected peptide for immunogenicity and overpresentation in the tumor by comparison with the corresponding normal tissue; (c) identifying at least one peptide in the patient. A step of selecting from the reservoir associated with the tumor-related peptide that has been identified; (d) optionally, a step of selecting at least one peptide newly identified in (a) and confirming its immunogenicity. Identified by.

In one exemplary embodiment, the peptides included in the vaccine are (a) a step of identifying a tumor-related peptide (TUMAP) presented by a tumor sample from an individual patient; (b) novel in (a). It is identified by a step of selecting at least one peptide identified and confirming its immunogenicity.

Once a peptide has been selected for a personalized peptide-based vaccine, the vaccine is manufactured. The vaccine is preferably a liquid formulation consisting of individual peptides dissolved in 20-40% DMSO, preferably about 30-35% DMSO, such as about 33% DMSO.

Each peptide included in the product is dissolved in DMSO. The concentration of the single peptide solution should be selected according to the number of peptides contained in the product. Equal volumes of single peptide DMSO solutions are mixed to give a solution containing all the peptides contained in the product at a concentration of about 2.5 mg / ml per peptide. The mixed solution is then diluted 1: 3 with water for injection to give a concentration of 0.826 mg / ml per peptide in 33% DMSO. The diluted solution is filtered through a 0.22 μm sterile filter. Obtain the final bulk solution.

Fill the vial with the final bulk solution and store at -20 ° C until use. One vial contains 700 μL of solution containing 0.578 mg of each peptide. Of this, 500 μL (approximately 400 μg per peptide) is applied for intradermal injection.

In addition to being useful for treating cancer, the peptides of the invention are also useful as diagnostic methods. Since the peptides were produced from the CRC and these peptides were determined to be absent or present at lower levels in normal tissue, these peptides can be used to diagnose the presence of cancer.

The presence of the patented peptide on a tissue biopsy in a blood sample may assist a pathologist in diagnosing cancer. Detection of certain peptides by means of antibodies, mass spectrometry or other methods known in the art can inform pathologists that the tissue sample is malignant or inflammatory or generally pathological, or of CRC. It can be used as a biomarker. The presence of peptide groups may allow classification or subclassification of diseased tissues.

Detection of peptides on affected tissue specimens allows the benefits of treatments involving the immune system to be determined, especially when T lymphocytes are known or predicted to be involved in the mechanism of action. Loss of MHC expression is a well-explained mechanism by which infected malignancies escape immune surveillance. Therefore, the presence of peptides indicates that this mechanism is not utilized by the cells analyzed.

The peptides of the invention may be used to analyze lymphocyte responses to these peptides, such as T cell responses or antibody responses to peptides complexed with peptides or MHC molecules. These lymphocyte responses can be used as prognostic markers to determine further stages of treatment. These responses can also be used as surrogate response markers in immunotherapeutic approaches aimed at inducing lymphocyte responses by different means, such as vaccination of proteins, nucleic acids, self-materials, and adopted lymphocyte immunotransmission. In the setting of gene therapy, the lymphocyte response to the peptide may be considered in the evaluation of side effects. Monitoring of lymphocyte response may also be a useful tool for follow-up of transplant therapy, such as detection of graft-versus-host disease and host-versus-graft disease.

The present invention will be described herein with reference to the accompanying drawings in the following examples illustrating preferred embodiments thereof, but are not limited thereto. For the purposes of the present invention, all references cited herein are incorporated by reference in their entirety.

It shows overpresentation of various peptides in normal tissue (white bar) and CRC (black bar). FIG. 1A: Gene symbol: ZNF679, ZNF716, SAPCD2, Peptide: ALIKQLFEA (SEQ ID NO: 1), tissue left to right: 1 adipose tissue, 3 adrenal glands, 6 arteries, 3 bone marrows, 7 brains, 3 breasts, 1 nerve (nerv) , 1 ovary, 8 esophagus, 2 bile sac, 5 heart, 16 kidney, 21 liver, 46 lung, 3 lymph node, 4 leukocyte sample, 3 ovary, 7 pancreas, 4 peripheral nerve, 1 peritoneum, 1 pituitary gland, 2 placenta , 3 peritoneum, 1 prostate, 2 salivary glands, 4 skeletal muscles, 4 skins, 2 small intestines, 4 spleen, 7 stomachs, 4 testicles, 2 thoracic glands, 3 thyroid glands, 1 trachea, 1 urinary tract, 3 bladder, 2 uterus, 2 Veins, 13 colons, 6 straight muscles, 24 CRC. Peptides are further detected on 9/99 lung cancer, 2/28 brain cancer, 4/20 ovarian cancer, 1/45 gastric cancer, 1/33 prostate cancer, and 2/15 esophageal cancer (not shown). Was done. It shows overpresentation of various peptides in normal tissue (white bar) and CRC (black bar). FIG. 1B: Gene symbol: BRCA2, Peptide: KQFEGTVII (SEQ ID NO: 138), Tissue left to right: 1 fatty tissue, 3 adrenal, 6 arteries, 3 bone marrow, 7 brain, 3 breast, 1 nerve (nerv), 1 ovary, 8 esophagus, 2 bile sac, 5 heart, 16 kidney, 21 liver, 46 lung, 3 lymph node, 4 leukocyte sample, 3 ovary, 7 pancreas, 4 peripheral nerve, 1 peritoneum, 1 pituitary gland, 2 placenta, 3 thoracic membrane, 1 prostate, 2 saliva glands, 4 skeletal muscles, 4 skins, 2 small intestines, 4 spleen, 7 stomachs, 4 testis, 2 thoracic glands, 3 thyroid glands, 1 trachea, 1 urinary tract, 3 bladder, 2 ovaries, 2 veins, 13 colons , 6 straight lines, 24 CRC. Peptides were further detected on 1/15 esophageal cancer, 1/28 brain cancer, 1/45 gastric cancer, and 3/91 lung cancer (not shown). It shows overpresentation of various peptides in normal tissue (white bar) and CRC (black bar). FIG. 1C: Gene symbol: IL8, Peptide: KLAVALLAA (SEQ ID NO: 210), Tissue left to right: 1 fatty tissue, 3 adrenal glands, 6 arteries, 3 bone marrows, 7 brains, 3 breasts, 1 nerve (nerv), 1 ovary, 8 esophagus, 2 bile sac, 5 heart, 16 kidney, 21 liver, 46 lung, 3 lymph node, 4 leukocyte sample, 3 ovary, 7 pancreas, 4 peripheral nerve, 1 peritoneum, 1 pituitary gland, 2 placenta, 3 thoracic membrane, 1 prostate, 2 saliva glands, 4 skeletal muscles, 4 skins, 2 small intestines, 4 spleen, 7 stomachs, 4 testis, 2 thoracic glands, 3 thyroid glands, 1 trachea, 1 urinary tract, 3 bladder, 2 ovaries, 2 veins, 13 colons , 6 straight lines, 24 CRC. Peptides are used on 14/99 lung cancer, 1/18 kidney cancer, 2/28 brain cancer, 2/16 liver cancer, 1/20 ovarian cancer, 1/45 gastric cancer, and 3/15 esophageal cancer. Further detected (not shown). It shows overpresentation of various peptides in normal tissue (white bar) and CRC (black bar). FIG. 1D) Gene symbol: TMEM222, Peptide: LLYGKYVSV (SEQ ID NO: 31) Tissue Left to right: 3 pancreatic cell line, 3 skin cell line, 1 leukemia cell line, 0 normal tissue, 28 cancer tissue (2 brain cancer, 1 breast cancer, 1 colon cancer, 1 esophageal cancer, 2 kidney cancer, 1 leukemia, 5 liver cancer, 7 lung cancer, 5 ovarian cancer, 1 prostate cancer, 2 rectal cancer). The normal tissue panels tested were identical to FIGS. 1A-C. The discrepancy in tumor type between FIG. 1D and Table 4 may be due to the more stringent selection criteria applied in Table 4 (see Table 4 for details). All samples with detectable presentation of peptide Y are shown, regardless of over-presentation parameters and technical sample quality checks. It shows overpresentation of various peptides in normal tissue (white bar) and CRC (black bar). FIG. 1E: Gene symbol: ZNF679, ZNF716, SAPCD2, Peptide: ALIKQLFEA (SEQ ID NO: 1), Tissue left to right: 7 cancer cell lines, 1 primary cancer cell culture, 58 cancer tissues (5 brain cancers) 1, 1 breast cancer, 9 colon cancer, 1 colon rectal cancer, 3 esophageal cancer, 1 bile sac cancer, 2 leukemia, 15 lung cancer, 2 lymph node cancer, 1 myeloid cell cancer, 5 ovary cancer, 1 Prostate cancer, 4 rectal cancer, 1 skin cancer, 2 stomach cancer, 2 bladder cancer, 3 uterine cancer). The normal tissue panels tested were identical to FIGS. 1A-C. It shows overpresentation of various peptides in normal tissue (white bar) and CRC (black bar). FIG. 1F: F) Gene symbol: PLAGL2, Peptide: FLAELPGSLSL (SEQ ID NO: 6), Tissue left to right: 8 cancer cell lines, 1 primary cancer cell culture, 2 normal tissues (1 lymph node, 1 spleen) , 57 cancer tissues (1 bone marrow cancer, 1 breast cancer, 1 cecum cancer, 5 colon cancer, 2 esophageal cancer, 1 bile sac cancer, 3 leukemia, 2 liver cancer, 13 lung cancer, 8 lymph node cancer 1, myeloid cell cancer, 9 ovarian cancer, 2 rectal cancer, 1 skin cancer, 1 stomach cancer, 4 bladder cancer, 2 uterine cancer). The normal tissue panels tested were identical to FIGS. 1A-C. It shows overpresentation of various peptides in normal tissue (white bar) and CRC (black bar). FIG. 1G: Gene symbol: CYP2W1, Peptide: FLDANGHFV (SEQ ID NO: 23), Tissue left to right: 1 Primary cancer cell culture, 3 Normal tissue (3 placenta), 12 cancer tissues (5 colon cancer, 1) Esophageal cancer, 1 bile sac cancer, 2 rectal cancer, 3 stomach cancer). The normal tissue panels tested were identical to FIGS. 1A-C. It shows overpresentation of various peptides in normal tissue (white bar) and CRC (black bar). FIG. 1H: Gene symbol: CYP2W1, Peptide: GLIDEVMVL (SEQ ID NO: 22), Tissue left to right: 1 Normal tissue (1 stomach), 6 cancer tissues (3 colon cancer, 1 gallbladder cancer, 2 rectal cancer) .. The normal tissue panels tested were identical to FIGS. 1A-C. It shows overpresentation of various peptides in normal tissue (white bar) and CRC (black bar). FIG. 1I: gene symbol: AXIN2, peptide: ILDDHLSRV (SEQ ID NO: 9), tissue from left to right: 5 cancer tissues (1 cecal cancer, 1 colon cancer, 1 lung cancer, 2 rectal cancer). The normal tissue panels tested were identical to FIGS. 1A-C. It shows overpresentation of various peptides in normal tissue (white bar) and CRC (black bar). FIG. 1J: Gene symbol: RAD54B, Peptide: KLLAVIHEL (SEQ ID NO: 152), Tissue left to right: 3 cell lines, 2 normal tissues (1 lymph node, 1 spleen), 34 cancer tissues (1 breast cancer, 7 colon cancer) 1, 1 esophageal cancer, 1 bile sac cancer, 1 kidney cancer, 8 lung cancer, 4 lymph node cancer, 1 myeloid cell cancer, 4 ovarian cancer, 1 pancreatic cancer, 1 rectal cancer, 3 skin Hmm, 1 bladder cancer). The normal tissue panels tested were identical to FIGS. 1A-C. It shows overpresentation of various peptides in normal tissue (white bar) and CRC (black bar). Figure 1K: Gene symbol: ECT2, Peptide: SLVQRVETI (SEQ ID NO: 142), Tissue left to right: 5 cell lines, 1 primary culture, 47 cancer tissues (2 bile duct cancer, 2 breast cancer, 1 cecum cancer, 7 colons) Cancer, 3 esophagus cancer, 3 bile sac cancer, 1 kidney cancer, 2 liver cancer, 10 lung cancer, 2 lymph node cancer, 4 ovary cancer, 1 pancreatic cancer, 2 rectal cancer, 2 skin Hmm, 1 stomach cancer, 2 bladder cancer, 2 uterine cancer). The normal tissue panels tested were identical to FIGS. 1A-C. It shows overpresentation of various peptides in normal tissue (white bar) and CRC (black bar). Figure 1L: Gene symbol: MMP12, Peptide: KIQEMQHFL (SEQ ID NO: 192), Tissue left-to-right: Primary culture, 44 cancer tissues (5 colon cancer, 1 esophageal cancer, 1 bile sac cancer, 1 head and neck) Hmm, 30 lung cancer, 1 lymph node cancer, 1 rectal cancer, 1 stomach cancer, 1 testicular cancer, 1 bladder cancer, 1 uterine cancer). The normal tissue panels tested were identical to FIGS. 1A-C. It shows overpresentation of various peptides in normal tissue (white bar) and CRC (black bar). FIG. 1M: Gene symbol: COL6A3, Peptide: FLLDGSANV (SEQ ID NO: 212), Tissue left to right: 3 cell lines, 2 normal tissues (1 placenta, 1 spleen), 146 cancer tissues (4 bile duct cancer, 13 breast cancer, 1 cul-de-sac cancer, 8 colon cancer, 1 colon rectal cancer, 6 esophageal cancer, 5 bile sac cancer, 5 head and neck cancer, 2 kidney cancer, 1 liver cancer, 62 lung cancer, 2 lymph node cancer , 9 ovarian cancer, 7 pancreatic cancer, 3 rectal cancer, 4 skin cancer, 5 stomach cancer, 5 bladder cancer, 3 uterine cancer). The normal tissue panels tested were identical to FIGS. 1A-C. An exemplary expression profile (normal) of the gene of origin of the invention, highly overexpressed or exclusively expressed in CRC in a panel of normal tissue (white bar) and 10 CRC samples (black bar). Relative expression compared to colon and rectum) is shown. Tissues From left to right: Adrenal, arteries, bone marrow, brain (whole), breast, colon, esophagus, heart, kidneys (triple), leukocytes, liver, lungs, lymph nodes, ovaries, pancreas, placenta, prostate, salivary glands, skeleton Muscle, skin, small intestine, spleen, stomach, testis, thoracic gland, thyroid, bladder, cervix, uterus, veins, 3 normal colon samples, 10 CRC samples. FIG. 2A, CCNB1; Same as above Figure 2B, CDK1; Same as above Figure 2C, CHMP5. An exemplary expression profile (normal) of the gene of origin of the invention, highly overexpressed or exclusively expressed in CRC in a panel of normal tissue (white bar) and 20 CRC samples (black bar). Relative expression compared to colon and rectum) is shown. Tissue left to right: 6 arteries, 2 blood cells, 2 brains, 1 heart, 2 livers, 3 lungs, 2 veins, 1 fat tissue, 1 thymus, 5 bone marrow, 1 cartilage, 1 colon, 1 esophagus, 2 eyes, 2 Gallbladder, 2 salivary glands, 1 kidney, 6 lymph nodes, 4 pancreas, 2 peripheral nerves, 2 pituitary gland, 1 rectum, 2 skeletal muscle, 1 skin, 1 small intestine, 1 spleen, 1 stomach, 1 thyroid, 7 trachea, 1 Gallbladder, 1 breast, 5 ovary, 5 placenta, 1 pancreas, 1 testicle, 1 thymus, 1 uterus, 20 CRC sample. FIG. 2D: ECT2. Shown exemplary immunogenicity data: Flow cytometry results after peptide-specific multimer staining. Shown are exemplary results of a healthy HLA-A ^* 02 + donor peptide-specific in vitro CD8 + T cell response. CD8 + T cells complex with SEQ ID NO: 22 peptide (A, left panel), SEQ ID NO: 9 peptide (B, left panel) or SEQ ID NO: 142 peptide (C, left panel), respectively, anti-CD28mAb and HLA-A ^*. Initial stimulation was performed using an artificial APC coated with 02. After 3 cycles of stimulation ^{, peptide reactivity by 2D multimer staining with A *} 02 / SEQ ID NO: 22 (A), A ^* 02 / SEQ ID NO: 9 (B) or A ^* 02 / SEQ ID NO: 142 (C). Cell detection was performed. The right panel (A, B, and C) shows counterstains of cells stimulated with ^{an unrelated A * 02 / peptide complex.} Surviving single cells were gated for CD8 + lymphocytes. Boolean gates helped eliminate false positive events detected by multimers specific for different peptides. The frequency of specific multimers + cells in CD8 + lymphocytes is shown. Same as above

Example 1
Identification and Quantification of Tumor-Related Peptides Presented on Cell Surface Tissue Samples Patient tumor tissues were obtained from the Universal Hospital of Tubingen.

Normal tissues include Asterand, Detroit, USA and Royston, Herts, UK; Bio-Options Inc, CA, USA; BioService, Beltsville, MD, USA; Capital BioScience Inc, Rockville. , Glendale, CA, USA; Tissue Solutions Ltd, Glasgow, Scotland, UK; University Hospital of Geneva; University Hospital of Heidelberg; Kyoto Prefectural University of Medicine (KPUM); University Hospital Munich; ProteoGenex Inc. , Culver City, CA, USA; obtained from the University Hospital of Tübingen. Consent based on all patient announcements was obtained prior to surgery or autopsy. Tissues were shock-frozen immediately after excision and stored below −70 ° C. until isolation of TUMAP.

^{Isolation of HLA Peptides from Tissue Samples HLA peptide reservoirs from shock-frozen tissue samples are HLA-A *} 02-specific according to a slightly modified protocol (Falk et al., 1991; Seeger et al., 1999). Antibodies BB7.2, HLA-A, -B, -C-specific antibodies W6 / 32, CNBr-activated Sepharose, acid treatment, and ultrafiltration were used to obtain from solid tissue by immunoprecipitation.

Mass Spectrometry The resulting HLA peptide reservoirs are separated according to their hydrophobicity by reverse phase chromatography (nanoAcquiity UPLC system, Waters) and placed in LTQ-velos and watersion hybrid mass spectrometers equipped with an ESI source. Eluted peptides were analyzed. Peptide reservoirs were inserted directly onto an analytical fusion silica microcapillary column (75 μm inner diameter x 250 mm) packed with 1.7 μm C18 reverse phase material (Waters) at a flow rate of 400 nL / min. Subsequently, peptides were separated using a two-step 180-minute two-component gradient of B from 10% to 33% at a flow rate of 300 nL / min. The gradient consisted of solvent A (0.1% formic acid in water) and solvent B (0.1% formic acid in acetonitrile). Gold-coated glass capillaries (PicoTip, New Objective) were used for introduction into the nanoESI source. The LTQ-Orbitrap mass spectrometer was operated in data-dependent mode using the TOP5 strategy. Briefly, a scan cycle is initiated with a high mass accuracy complete scan within Orbitrap (R = 30000), which is also an MS / MS scan of the five most abundant precursor ions in Orbitrap (R = 7500). Followed by, the previously selected ions were dynamically eliminated. The tandem mass spectrum was interpreted by SEQ and additional manual adjustment. The identified peptide sequences were confirmed by comparison of the generated native peptide fragmentation pattern with the fragmentation pattern of synthetic reference peptides of the same sequence.

Unlabeled relative LC-MS quantification was performed by ion counting, ie by extraction and analysis of LC-MS properties (Mueller et al., 207). The method assumes that the LC-MS signal area of the peptide correlates with its abundance in the sample. The extracted properties were further processed by charge state deconvolution and residence time alignment (Mueller et al., 2008; Sturm et al., 2008). Finally, all LC-MS properties were cross-referenced with the sequence identification results to combine the quantitative data of the different samples with the tissue-to-peptide presentation profile. Quantitative data were normalized by a two-step method according to central tendency, taking into account variations within technical and biological repeat tests. In this way, each identified peptide can be associated with quantitative data, allowing for relative quantification between the sample and the tissue. In addition, all quantitative data obtained for peptide candidates were manually inspected to ensure data integrity and confirm the accuracy of automated analysis. Presentation profiles were calculated for each peptide and showed mean sample presentation and repeat test variability. The profile juxtaposes the CRC sample to the baseline of the normal tissue sample. The presentation profile of the exemplary overpresented peptide is shown in FIG. The presentation scores for representative peptides are shown in Table 8.

Table 8: Presented scores. The table is highly over-presented on the tumor compared to the normal tissue panel (+++), highly over-presented on the tumor compared to the normal tissue panel (++), and with the normal tissue panel. List peptides that are over-presented (+) on tumors in comparison.

Example 2
Gene expression profiling encoding the peptides of the invention Overpresentation or specific presentation of peptides on tumor cells compared to normal cells is sufficient for their usefulness in immunotherapy, and some peptides are proteins of their origin. Is tumor-specific, even though it is also present in normal tissues. Nonetheless, mRNA expression profiling can increase the level of safety in the selection of peptide targets for immunotherapy. In particular, for high-risk therapeutic options such as affinity matured TCR, the ideal target peptide is derived from a protein that is unique to the tumor and is not found on normal tissues.

RNA Origin and Preparation Surgically removed tissue specimens were provided as described above after a notice-based consent form was obtained from each patient (see Example 1). Tumor tissue specimens were snap-frozen immediately after surgery and then homogenized under liquid nitrogen using a mortar and pestle. Total RNA was prepared from these samples using TRI reagents (Ambion, Darmstadt, Germany), followed by purification with RNeasy (QIAGEN, Hilden, Germany); both methods were performed according to the manufacturer's protocol.

Total RNA from healthy human tissue was commercially available (Ambion, Huntingdon, UK; Clontech, Heidelberg, Germany; Stratagene, Amsterdam, Netherlands; BioChain, Hayward, CA, USA). RNA from several (2-123) individuals was mixed so that RNA from each individual was equally weighted.

The quality and quantity of all RNA samples were assessed on an Agilent 2100 Bioanalyzer (Agilent, Waldbronn, Germany) using the RNA 6000 Pico LabChip kit (Aglent).

Microarray Experiments Gene expression analysis of all tumor and normal tissue RNA samples was performed by Affymetrix Human Genome (HG) U133A or HG-U133 Plus 2.0 oligonucleotide microarrays (Affymetrix, Santa Clara, CA, USA). All steps were performed according to the Affymetrix manual. Briefly, double-stranded cDNA from 5-8 μg of total RNA using SuperScript RTII (Invitrogen) and oligo dT-T7 primers (MWG Biotech, Ebersberg, Germany) as described in the manual. Synthesized. In vitro transcription can be performed using the BioAryHigh Yield RNA Transtript Laboring Kit (ENZO Diagnostics, Inc., Farmingdale, NY, USA) for the U133A array, or from the Gent after the U133 Plus Followed by cRNA fragmentation, hybridization, and staining with streptavidin-phycoerythrin and biotinylated anti-streptavidin antibodies (Molecular Probes, Leiden, Netherlands). Images were scanned with an Agilent 2500A GeneArray Scanner (U133A) or Affymetrix Gene-Chip Scanner 3000 (U133 Plus 2.0) and the data was analyzed by GCOS software (Affymetrix) using default settings for all parameters. For normalization, 100 housekeeping genes provided by Affymetrix were used. Relative expression values were calculated from the signal log ratio given by the software and normal kidney samples were set to 1.0 at their discretion. A representative expression profile of the gene of origin of the invention, which is highly overexpressed or exclusively expressed in CRC, is shown in FIG. Expression scores for additional exemplary genes are shown in Table 9.

Table 9: Expression score. The table is very highly overexpressed in tumors compared to normal tissue panels (+++), highly overexpressed in tumors compared to normal tissue panels (++), and compared to normal tissue panels. Lists gene-derived peptides that are overexpressed in tumors (+).

Example 3
In Vitro Immunogenicity of MHC Class I Presenting Peptides To obtain information on the immunogenicity of TUMAPs of the invention, we present artificial antigen presenting cells (aAPC) loaded with peptide / MHC complexes and anti-CD28 antibodies. ) Was used to perform the study using an in vitro T cell priming assay based on repeated stimulation of CD8 + T cells. In this way, we have shown the immunogenicity of the 22 HLA-A ^* 0201-binding TUMAPs of the invention so far, in which these peptides are CD8 + precursor T cells to humans. It was demonstrated that it is a T cell epitope present in (Table 10).

In vitro priming of CD8 + T cells To perform in vitro stimulation with artificial antigen-presenting cells loaded with peptide MHC complex (pMHC) and anti-CD28 antibodies, we first, after consent based on the announcement, ^{Fresh HLA-A *} 02 leukocyte-removed product isolated from fresh HLA-A * 02 leukocyte-removed product through positive selection using CD8 microbeads (Miltenyi Biotec, Bergisch-Gradbach, Germany) from healthy donors obtained from Universality clinics Mannheim, Germany. ..

PBMCs and isolated CD8 + lymphocytes or PBMCs are 10% heat-inactivated human AB serum (PAN-Biotech, Aidenbach, Germany), 100 U / ml penicillin / 100 μg / ml streptomycin (Cambrex, Collogne, Germany), 1 mM pyruvate. The cells were cultured in T cell medium (TCM) consisting of RPMI-Glutamax (Invitrogen, Karlsruhe, Germany) supplemented with sodium (CC Pro, Oberdora, Germany) and 20 μg / ml gentamicin (Cambrex) until use. 2.5 ng / ml IL-7 (PromoCell, Heidelberg, Germany) and 10 U / ml IL-2 (Novartis Pharma, Nuremberg, Germany) were also added to the TCM at this stage.

Generation, T cell stimulation, and reading of pMHC / anti-CD28 coated beads uses 4 different pMHC molecules per stimulation condition and 8 different pMHC molecules per reading condition within a highly defined in vitro system. And carried out.

Purified co-stimulated mouse IgG2a anti-human CD28 Ab9.3 (Jung et al., 1987) is chemically biotin using sulfo-N-hydroxysuccinimide biotin as recommended by the manufacturer (Perbio, Bonn, Germany). It became. The beads used were streptavidin-coated polystyrene particles (Bangs Laboratories, Illinois, USA) with a diameter of 5.6 μm.

The pMHCs used for positive and negative control stimuli were A ^* 0201 / MLA-001 (peptide ELAGIGILTV (SEQ ID NO: 266) from modified Melan-A / MART-1) and A ^* 0201 / DDX5-, respectively. It was 001 (YLLPAIVHI derived from DDX5, SEQ ID NO: 267).

In the presence of 4 × 12.5 ng of different biotin pMHC, 800,000 beads / 200 μl were coated in a 96-well plate, washed and subsequently added 600 ng of biotin anti-CD28 in a volume of 200 μl. .. In 200 μl TCM supplemented with 5 ng / ml IL-12 (PromoCell), 1 × 10 ⁶ CD8 + T cells were co-incubated with 2 × 10 ⁵ wash-coated beads at 37 ° C. for 3 days. Stimulation was initiated in a 96-well plate. Half of the medium was then replaced with fresh TCM supplemented with 80 U / ml IL-2 and culture was continued at 37 ° C. for 4 days. This stimulation cycle was performed a total of 3 times. In pMHC multimer readings using 8 different pMHC molecules per condition, with minor modifications including conjugation to 5 different fluorochromes, as previously described (Andersen et al., 2012). A two-dimensional combinatorial coding approach was used. Finally, multimer analysis was performed by staining cells with Live / dead near-infrared dyes (Invitrogen, Karlsruhe, Germany), CD8-FITC antibody clone SK1 (BD, Heidelberg, Germany), and fluorescent pMHC multimers. The analysis used a BD LSRII SORP blood cell counter equipped with a suitable laser and filter. Peptide-specific cells were calculated as a percentage of total CD8 + cells. Multimer analysis was evaluated using FlowJo software (Tree Star, Oregon, USA). In vitro initial stimulation of specific multimers + CD8 + lymphocytes was detected by comparison with negative control stimulation. If at least one evaluable in vitro stimulation well in a healthy donor is found to contain a specific CD8 + T cell line after in vitro stimulation, the immunogenicity of a given antigen has been detected (ie,). This well contained at least 1% specific multimer + in CD8 + T cells, and the percentage of specific multimer + cells was at least 10 times the median negative control stimulus).

In Vitroimmunogenicity of CRC Peptides To test HLA class I peptides, the generation of peptide-specific T cell lines could demonstrate in vitro immunogenicity. Exemplary flow cytometric results after TUMAP-specific multimer staining of one peptide of the invention are shown in FIG. 3 with a corresponding negative control. The results of the two peptides from the present invention are summarized in Table 10A.

Table 10A: In Vitroimmunogenicity of HLA Class I Peptides of the Invention Illustrative results of in vitro immunogenicity experiments of the peptides of the invention performed by the applicant. <20% = +; 20% --49% = ++; 50% --69% = +++;> = 70% = ++++

Table 10B: Additional data on the in vitro immunogenicity of the HLA class I peptides of the invention.
This is a representative result of an in vitro immunogenicity experiment on the ^{HLA-A *} 02 restrictive peptide of the present invention carried out by the applicant. The results of in vitro immunogenicity experiments are shown. Percentages of positive wells and donors (within evaluable) are summarized as shown <20% = +; 20%-49% = ++; 50%-69% = +++;> = 70% = ++++

Example 4
Peptide Synthesis All peptides were synthesized using standard, well-established solid phase peptide synthesis using the Fmoc strategy. The identity and purity of the individual peptides were determined by mass spectrometry and analytical RP-HPLC. The peptide was obtained as a lyophilized product (trifluoroacetic acid salt) of white to grayish white with a purity of> 50%. All TUMAPs are preferably administered as trifluoroacetic acid salts or acetates, and other salt forms are also possible.

Example 5
MHC Binding Assays Candidate peptides for T cell-based therapies according to the invention were further tested for their MHC binding capacity (affinity). Individual peptide-MHC complexes were generated by UV ligand exchange, and UV sensitive peptides were cleaved upon UV irradiation and replaced with the peptide of interest to be analyzed. Only peptide candidates that can effectively bind and stabilize peptide-accepting MHC molecules prevent separation of the MHC complex. To determine the yield of the exchange reaction, an ELISA based on the detection of the light chain (β2m) of the stabilized MHC complex was performed. The assay was performed by Rodenko et al. (Rodonko et al., 2006).

A 96-well MAXISorp plate (NUNC) was coated overnight with 2 μg / ml streptavidin in PBS at room temperature, washed 4 times and blocked at 37 ° C. for 1 hour in 2% BSA containing block buffer. The refolded HLA-A ^* 020102: 01 / MLA-001 monomer served as a reference material covering the range of 15-500 ng / ml. The peptide-MHC monomer of the UV exchange reaction was diluted 100-fold with block buffer. Samples were incubated at 37 ° C. for 1 hour, washed 4 times, incubated with 2 μg / ml HRP-binding anti-β2 m for 1 hour at 37 ° C., washed again and detected in TMB solution stopped with NH2SO4. Absorption was measured at 450 nm. Candidate peptides that exhibit high exchange yields (preferably greater than 50%, most preferably greater than 75%) for the production and production of antibodies or fragments thereof, and / or T cell receptors or fragments thereof. It is generally preferred because it exhibits sufficient binding activity to MHC molecules and prevents separation of the MHC complex.

Table 11: MHC class I binding score. Binding of HLA class I restrictive peptides to HLA-A ^* 02: 01 varied with peptide exchange yields: > 10% = +; > 20% = ++; > 50 = +++; > 75% = ++++; J = Phosphoserine

Example 6
Absolute Quantification of Tumor-Related Peptides Presented on Cell Surface The production of antibodies and / or binders such as TCRs is a painstaking process and may only be performed on a few selected targets. For tumor-related and specific peptides, selection criteria include, but are not limited to, the exclusivity of presentation and the density of peptides presented on the cell surface. In addition to the peptide isolation and relative quantification described in Example 1, we analyzed the absolute peptide copy number per cell as described in Patent x (patentx). Quantification of TUMAP copies per cell in solid tumor samples requires absolute quantification of isolated TUMAPs, efficiency of TUMAP isolation, and cell counting of tissue samples analyzed. An overview of our experimental approach is shown in FIG. 4, and the experimental procedure is described below.

Peptide Quantification by Nano LC-MS / MS For accurate quantification of peptides by mass spectrometry, a calibration curve was prepared for each peptide using the internal standard method. The internal standard is a double isotope-labeled variant of each peptide, i.e., two isotope-labeled amino acids were included in TUMAP synthesis. It differs only in its mass from tumor-related peptides, but does not show any difference in other physicochemical properties (Anderson et al., 2012). An internal standard was added to each MS sample to normalize all MS signals to the internal standard MS signals and level out potential technical variations between MS experiments.

Calibration curves were made in at least three different matrices, ie in HLA peptide eluates from natural samples similar to routine MS samples, and each preparation was measured in a series of MS tests. For evaluation, the MS signal was normalized to the internal standard signal and the calibration curve was calculated by logistic regression.

For the quantification of tumor-related peptides from tissue samples, an internal standard was also added to each sample; MS signals were normalized to the internal standard and quantified using a peptide calibration curve.

Efficiency of Peptide / MHC Isolation As with any protein purification process, isolation of a protein from a tissue sample involves some loss of the protein of interest. Peptide / MHC complexes were generated for all TUMAPs selected for absolute quantification to determine the efficiency of TUMAP isolation. A single isotope-labeled version of TUMAP was used, i.e., one isotope-labeled amino acid was included in TUMAP synthesis so that the addition could be distinguished from the native peptide / MHC complex. These complexes are added to freshly prepared tissue lysates, i.e., as early as possible in the TUMAP isolation procedure, and then in the following affinity purifications, such as the natural peptide / MHC complex. Was captured in. Therefore, measuring the recovery of single-labeled TUMAP allows conclusions regarding the isolation efficiency of individual native TUMAPs.

Isolation efficiencies were analyzed on a small number of samples and were comparable between these tissue samples. In contrast, isolation efficiencies vary among individual peptides. This suggests that isolation efficiency is determined only in a limited number of tissue samples, but may be extrapolated to any other tissue specimen. However, each TUMAP needs to be analyzed individually, as isolation efficiency may not be extrapolated from one peptide to another.

Cell Counting in Solid Frozen Tissues To measure the cell number of tissue samples used for absolute peptide quantification, we applied DNA content analysis. This method can be applied to a wide range of samples from different origins, and most importantly to frozen samples (Alcoser et al., 2011; Forsey and Chaudhuri, 2009; Silva et al., 2013). During the peptide isolation protocol, the tissue sample is processed into a homogeneous lysate and then a small lysate aliquot is removed. The aliquot is divided into three and the DNA is isolated from it (QiaAmp DNA Mini Kit, Qiagen, Hilden, Germany). A fluorescence-based DNA quantification assay (Qubit dsDNA HS Assay Kit, Life Technologies, Darmstadt, Germany) is used to quantify the total DNA content from each DNA isolation in at least two repeat tests.

To calculate cell numbers, a DNA standard curve was created from an aliquot of single healthy blood cells with a set of defined cell numbers. The standard curve is used to calculate the total cell content from the total DNA content from each DNA isolate. Extrapolate the average total cell number of the tissue sample used for peptide isolation, taking into account the volume of known lysate aliquots and the volume of total lysate.

Number of copies per peptide cell Using the data from the above experiments, we divided the total amount of peptide in the sample by the total number of cells, followed by the isolation efficiency to TUMAP per cell. The number of copies was calculated. The cell copy numbers of the selected peptides are shown in Table 12.

Table 12: Absolute number of copies. The table lists the results of absolute peptide quantification in NSCLC tumor samples. The median number of copies per cell is shown for each peptide: <100 = +;> = 100 = ++;> = 1,000 +++;> = 10,000 = ++++. The number of samples available for evaluable high quality MS data is shown.

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Claims (15)

A peptide consisting of the amino acid sequence shown in SEQ ID NO: 22, with the ability to bind to the peptide major histocompatibility complex (MHC) class I partial child, peptide, or a pharmaceutically acceptable salt thereof,. When bound to the MHC molecules, it is possible to be recognized by C D8T cells, peptide according to claim 1 or a pharmaceutically acceptable salt thereof. A modified and / or non-peptide binding peptide, or a pharmaceutically acceptable salt thereof, wherein the peptide according to claim 1 or 2 or a pharmaceutically acceptable salt thereof is modified and / or contains a non-peptide bond. .. A fusion protein comprising the N-terminal amino acid of the HLA-DR antigen-related invariant chain (Ii) and the peptide according to any one of claims 1 to 3 or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable salt thereof. Acceptable salt. A T cell receptor that is a soluble or membrane-bound T cell receptor that is reactive with an HLA ligand bound to an MHC molecule, wherein the ligand is the peptide defined in claim 1, soluble or soluble or A membrane-bound T cell receptor that is reactive with an HLA ligand bound to an MHC molecule and said the ligand is a T cell receptor that is the peptide defined in claim 1, said T. A T cell receptor in which the cell receptor is provided as a soluble molecule and possesses additional effector functions selected from immunostimulatory domains or toxins. The peptide according to claim 1 or 2, the nucleic acid encoding the T cell receptor according to claim 5, or an expression vector capable of expressing the nucleic acid, or the peptide according to claim 1 or 2. 5. The nucleic acid encoding the T cell receptor according to 5, or an expression vector capable of expressing the nucleic acid, wherein the nucleic acid or the expression vector is bound to a heterologous promoter sequence. The peptide according to claim 1 or 2, or the nucleic acid or expression vector according to claim 6, or comprising the peptide according to claim 1 or 2, or the nucleic acid or expression vector according to claim 6. A recombinant host cell comprising the above, wherein the host cell is an antigen-presenting cell, or contains the peptide according to claim 1 or 2, or the nucleic acid or expression vector according to claim 6. A recombinant host cell comprising a nucleic acid encoding the T cell receptor, wherein the host cell is a T cell or an NK cell.
Recombinant host cell. A step of culturing a host cell according to claim 7, wherein the peptide according to claim 1 or 2 is presented, or the nucleic acid according to claim 6 is expressed, or the expression vector according to claim 6 is provided. The method for producing a peptide according to claim 1 or 2, comprising the step of isolating the peptide from the host cell or a culture solution thereof. Activating T cells in an antigen-specific manner to an antigen-loaded human class I MHC molecule expressed on the surface of a suitable antigen-presenting cell or on the surface of an artificial construct that mimics the antigen-presenting cell. An in vitro method for producing activated T lymphocytes, comprising the step of in vitro contacting the antigen for a sufficient period of time, wherein the antigen is the peptide according to claim 1 or 2. Activated T lymphocytes produced by the method of claim 9, which selectively recognizes cells presenting the peptide of claim 1 or 2. The target cell killing agent comprising the activated T lymphocyte according to claim 10, wherein the target cell presents the peptide according to claim 1 or 2. The peptide according to claim 1 or 2, or a pharmaceutically acceptable salt thereof, the fusion protein according to claim 4, or a pharmaceutically acceptable salt thereof, the T cell receptor according to claim 5, the claim. Pharmaceutically acceptable with at least one active ingredient selected from the group consisting of the nucleic acid or expression vector of claim 6, the recombinant host cell of claim 7, or the activated T lymphocytes of claim 10. A pharmaceutical composition comprising a possible carrier. The peptide according to any one of claims 1 to 3 or a pharmaceutically acceptable salt thereof, the fusion protein according to claim 4 or a pharmaceutically acceptable salt thereof, according to claim. 5. The T cell receptor according to claim 5, the nucleic acid or expression vector according to claim 6, the host cell according to claim 7, the activated T lymphocyte according to claim 10, or the drug according to claim 12. A drug comprising a composition. The peptide according to any one of claims 1 to 3 or a pharmaceutically acceptable salt thereof, the fusion protein according to claim 4, or the fusion protein according to claim 4, in the manufacture of a drug for treating cancer or for treating cancer. The pharmaceutically acceptable salt thereof, the T cell receptor according to claim 5, the nucleic acid or expression vector according to claim 6, the host cell according to claim 7, and the activated T lymph according to claim 10. The peptide according to any one of claims 1 to 3 or a pharmaceutical thereof in the use of a sphere or the pharmaceutical composition according to claim 12, or in the manufacture of a drug for treating cancer or for treating cancer. 4. The fusion protein according to claim 4 or a pharmaceutically acceptable salt thereof, the T cell receptor according to claim 5, the nucleic acid or expression vector according to claim 6, claim 7. The use of the host cell according to claim 10, the activated T lymphocyte according to claim 10 or the pharmaceutical composition according to claim 12, wherein the agent is for vaccine or cell therapy, is used, or treats cancer. The peptide according to any one of claims 1 to 3 or a pharmaceutically acceptable salt thereof, the fusion protein according to claim 4, or the pharmaceutically acceptable salt thereof. Acceptable salt, T cell receptor according to claim 5, nucleic acid or expression vector according to claim 6, host cell according to claim 7, activated T lymphocyte or claim 10 according to claim 10. use of a pharmaceutical composition according to 12, wherein the cancer is lung cancer, brain cancer, liver cancer, kidney cancer, colorectal cancer, liver cancer, pancreatic cancer, prostate cancer , leukemia, breast cancer, Merkel cell carcinoma, melanoma, in is selected from the group of ovarian cancer, and esophageal cancer, use or manufacture or cancer therapeutic agent for treating cancer, claim 1 3. The peptide according to any one of 3 or a pharmaceutically acceptable salt thereof, the fusion protein according to claim 4 or a pharmaceutically acceptable salt thereof, the T cell receptor according to claim 5. The use of the nucleic acid or expression vector according to claim 6, the host cell according to claim 7, the activated T lymphocyte according to claim 10, or the pharmaceutical composition according to claim 12, wherein the drug is used. a vaccine or cell therapy, wherein the cancer is lung cancer, brain cancer, liver cancer, kidney cancer, colorectal cancer, liver cancer, pancreatic cancer, prostate cancer, leukemia, breast cancer, Used, selected from the group of Mercel cell cancer, melanoma, ovarian cancer, and esophageal cancer. (A) The peptide according to any one of claims 1 to 3 or a pharmaceutically acceptable salt thereof, the fusion protein according to claim 4 or a pharmaceutically acceptable salt thereof, according to claim 5. T cell receptor, the nucleic acid or expression vector according to claim 6, the host cell according to claim 7, or the pharmaceutical composition containing the activated T lymphocytes according to claim 10, in a solution or freeze-dried form. And (b) a second container containing a diluent or reconstitution solution for the lyophilized formulation;
A kit comprising: (a) the peptide according to any one of claims 1 to 3 or a pharmaceutically acceptable salt thereof, the fusion protein according to claim 4 or a pharmaceutically acceptable salt thereof. A pharmaceutical composition comprising a salt, the T cell receptor of claim 5, the nucleic acid or expression vector of claim 6, the host cell of claim 7, or the activated T lymphocytes of claim 10. A container comprising the material in solution or lyophilized form; and (b) a second container containing the diluent or reconstituted solution for the lyophilized formulation;
, Which further comprises at least one peptide selected from the group consisting of the amino acid sequences set forth in SEQ ID NOs: 1-21 and SEQ ID NOs: 23-191, or (a). The peptide according to any one of claims 1 to 3 or a pharmaceutically acceptable salt thereof, the fusion protein according to claim 4 or a pharmaceutically acceptable salt thereof, and the T cell according to claim 5. A pharmaceutical composition containing the receptor, the nucleic acid or expression vector of claim 6, the host cell of claim 7, or the activated T lymphocytes of claim 10, in solution or lyophilized form. Vessel; and (b) a second vessel containing a diluent or reconstituted solution for the lyophilized formulation;
A kit comprising: (i) buffer, (ii) diluent, (iii) filter, (iv) needle, or (V) further comprising one or more selected from a syringe. The kit, or (a) the peptide according to any one of claims 1 to 3 or a pharmaceutically acceptable salt thereof, the fusion protein according to claim 4 or a pharmaceutically acceptable salt thereof, claim. A solution or a pharmaceutical composition containing the T cell receptor according to claim 5, the nucleic acid or expression vector according to claim 6, the host cell according to claim 7, or the activated T lymphocyte according to claim 10. A container comprising the lyophilized form; and (b) a second container containing the diluent or reconstituted solution for the lyophilized formulation;
A kit comprising, further comprising at least one peptide selected from the group consisting of the amino acid sequences set forth in SEQ ID NOs: 1-21 and SEQ ID NOs: 23-191, (i) buffer. A kit further comprising one or more selected from (ii) diluent, (iii) filter, (iv) needle, or (V) syringe.

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